3
PROTEIN STRUCTURE
AND FUNCTION
Electron density map of the F1-ATPase associated w ith
a ring of 10 c-subunits from the F0 domain of ATP
synthase, a molecular machine that carries out the
synthesis of ATP in eubacteria, chloroplasts, and
mitochondria. [Courtesy of Andrew Leslie, M RC Laboratory of
M olecular Biology, Cambridge, UK.]
P
roteins, the working molecules of a cell, carry out the
program of activities encoded by genes. This program
requires the coordinated effort of many different types
of proteins, which first evolved as rudimentary molecules
that facilitated a limited number of chemical reactions. Gradually, many of these primitive proteins evolved into a wide
array of enzymes capable of catalyzing an incredible range of
intracellular and extracellular chemical reactions, with a
speed and specificity that is nearly impossible to attain in a
test tube. With the passage of time, other proteins acquired
specialized abilities and can be grouped into several broad
functional classes: structural proteins, which provide structural rigidity to the cell; transport proteins, which control the
flow of materials across cellular membranes; regulatory proteins, which act as sensors and switches to control protein
activity and gene function; signaling proteins, including cellsurface receptors and other proteins that transmit external
signals to the cell interior; and m otor proteins, which cause
motion.
A key to understanding the functional design of proteins
is the realization that many have “ moving” parts and are capable of transmitting various forces and energy in an orderly
fashion. H owever, several critical and complex cell
processes—synthesis of nucleic acids and proteins, signal
transduction, and photosynthesis—are carried out by huge
macromolecular assemblies sometimes referred to as m olecular m achines.
A fundamental goal of molecular cell biologists is to understand how cells carry out various processes essential for
life. A major contribution toward achieving this goal is the
identification of all of an organism’s proteins—that is, a list
of the parts that compose the cellular machinery. The compilation of such lists has become feasible in recent years with
the sequencing of entire genomes—complete sets of genes—
of more and more organisms. From a computer analysis of
OU TLIN E
3.1 Hierarchical Structure of Proteins
3.2 Folding, Modification, and Degradation
of Proteins
3.3 Enzymes and the Chemical Work of Cells
3.4 Molecular Motors and the Mechanical Work
of Cells
3.5 Common Mechanisms for Regulating Protein
Function
3.6 Purifying, Detecting, and Characterizing Proteins
59
60
CHAPTER 3 • Protein Structure and Function
genome sequences, researchers can deduce the number and
primary structure of the encoded proteins (Chapter 9). The
term proteome was coined to refer to the entire protein complement of an organism. For example, the proteome of the
yeast Saccharom yces cerevisiae consists of about 6000 different proteins; the human proteome is only about five times
as large, comprising about 32,000 different proteins. By
comparing protein sequences and structures, scientists can
classify many proteins in an organism’s proteome and deduce
their functions by homology with proteins of known function. Although the three-dimensional structures of relatively
few proteins are known, the function of a protein whose
structure has not been determined can often be inferred from
its interactions with other proteins, from the effects result(a)
M OLECULAR STRUCTURE
Prim ary (sequence)
Secondary (local folding)
Tertiary (long-range folding)
3.1 Hierarchical Structure of Proteins
Quaternary (m ultim eric organization)
Supram olecular (large-scale assem blies)
(b)
"off "
Regulation
Signaling
"on"
Structure
FUNCTION
M ovem ent
ing from genetically mutating it, from the biochemistry of the
complex to which it belongs, or from all three.
In this chapter, we begin our study of how the structure
of a protein gives rise to its function, a theme that recurs
throughout this book (Figure 3-1). The first section examines
how chains of amino acid building blocks are arranged and
the various higher-order folded forms that the chains assume.
The next section deals with special proteins that aid in the
folding of proteins, modifications that take place after the
protein chain has been synthesized, and mechanisms that degrade proteins. The third section focuses on proteins as catalysts and reviews the basic properties exhibited by all
enzymes. We then introduce molecular motors, which convert chemical energy into motion. The structure and function
of these functional classes of proteins and others are detailed
in numerous later chapters. Various mechanisms that cells
use to control the activity of proteins are covered next. The
chapter concludes with a section on commonly used techniques in the biologist’s tool kit for isolating proteins and
characterizing their properties.
Transport
Catalysis
A
B
▲ FIGU RE 3 -1 Overview of protein structure and function.
(a) The linear sequence of amino acids (primary structure) folds
into helices or sheets (secondary structure) w hich pack into a
globular or fibrous domain (tertiary structure). Some individual
proteins self-associate into complexes (quaternary structure) that
can consist of tens to hundreds of subunits (supramolecular
assemblies). (b) Proteins display functions that include catalysis of
chemical reactions (enzymes), flow of small molecules and ions
(transport), sensing and reaction to the environment (signaling),
control of protein activity (regulation), organization of the genome,
lipid bilayer membrane, and cytoplasm (structure), and generation
of force for movement (motor proteins). These functions and
others arise from specific binding interactions and conformational
changes in the structure of a properly folded protein.
Although constructed by the polymerization of only 20 different amino acids into linear chains, proteins carry out an
incredible array of diverse tasks. A protein chain folds into
a unique shape that is stabilized by noncovalent interactions
between regions in the linear sequence of amino acids. This
spatial organization of a protein—its shape in three dimensions—is a key to understanding its function. O nly when a
protein is in its correct three-dimensional structure, or conformation, is it able to function efficiently. A key concept in
understanding how proteins work is that function is derived
from three-dim ensional structure, and three-dim ensional
structure is specified by am ino acid sequence. H ere, we consider the structure of proteins at four levels of organization,
starting with their monomeric building blocks, the amino
acids.
The Primary Structure of a Protein Is Its Linear
Arrangement of Amino Acids
We reviewed the properties of the amino acids used in synthesizing proteins and their linkage by peptide bonds into linear chains in Chapter 2. The repeated amide N , ␣ carbon
(C ␣), and carbonyl C atoms of each amino acid residue form
the back bone of a protein molecule from which the various
side-chain groups project (Figure 3-2). As a consequence of
the peptide linkage, the backbone exhibits directionality because all the amino groups are located on the same side of
the C ␣ atoms. Thus one end of a protein has a free (unlinked)
amino group (the N -term inus) and the other end has a free
carboxyl group (the C-term inus). The sequence of a protein
chain is conventionally written with its N -terminal amino
acid on the left and its C-terminal amino acid on the right.
3.1 • Hierarchical Structure of Proteins
aa1
R
aa2
aa3
Peptide
bond
R
R
Peptide
bond
▲ FIGU RE 3 -2 Structure of a tripeptide. Peptide bonds
(yellow ) link the amide nitrogen atom (blue) of one amino acid
(aa) w ith the carbonyl carbon atom (gray) of an adjacent one in
the linear polymers know n as peptides or polypeptides,
depending on their length. Proteins are polypeptides that have
folded into a defined three-dimensional structure (conformation).
The side chains, or R groups (green), extending from the ␣
carbon atoms (black) of the amino acids composing a protein
largely determine its properties. At physiological pH values, the
terminal amino and carboxyl groups are ionized.
The primary structure of a protein is simply the linear
arrangement, or sequence, of the amino acid residues that
compose it. M any terms are used to denote the chains
formed by the polymerization of amino acids. A short chain
of amino acids linked by peptide bonds and having a defined
sequence is called a peptide; longer chains are referred to as
polypeptides. Peptides generally contain fewer than 20–30
amino acid residues, whereas polypeptides contain as many
as 4000 residues. We generally reserve the term protein for
a polypeptide (or for a complex of polypeptides) that has a
well-defined three-dimensional structure. It is implied that
proteins and peptides are the natural products of a cell.
The size of a protein or a polypeptide is reported as its
mass in daltons (a dalton is 1 atomic mass unit) or as its molecular weight (M W), which is a dimensionless number. For
example, a 10,000-MW protein has a mass of 10,000 daltons
(Da), or 10 kilodaltons (kDa). In the last section of this chapter, we will consider different methods for measuring the sizes
and other physical characteristics of proteins. The known and
predicted proteins encoded by the yeast genome have an average molecular weight of 52,728 and contain, on average,
466 amino acid residues. The average molecular weight of
amino acids in proteins is 113, taking into account their average relative abundance. This value can be used to estimate the
number of residues in a protein from its molecular weight or,
conversely, its molecular weight from the number of residues.
Secondary Structures Are the Core Elements
of Protein Architecture
The second level in the hierarchy of protein structure consists
of the various spatial arrangements resulting from the folding of localized parts of a polypeptide chain; these arrangements are referred to as secondary structures. A single
61
polypeptide may exhibit multiple types of secondary structure depending on its sequence. In the absence of stabilizing
noncovalent interactions, a polypeptide assumes a random coil structure. H owever, when stabilizing hydrogen bonds
form between certain residues, parts of the backbone fold
into one or more well-defined periodic structures: the alpha
(␣) helix, the beta () sheet, or a short U-shaped turn. In an
average protein, 60 percent of the polypeptide chain exist as
␣ helices and  sheets; the remainder of the molecule is in
random coils and turns. Thus, ␣ helices and  sheets are the
major internal supportive elements in proteins. In this section, we explore forces that favor the formation of secondary
structures. In later sections, we examine how these structures
can pack into larger arrays.
The ␣ Helix In a polypeptide segment folded into an ␣ helix,
the carbonyl oxygen atom of each peptide bond is hydrogenbonded to the amide hydrogen atom of the amino acid four
residues toward the C-terminus. This periodic arrangement of
bonds confers a directionality on the helix because all the
hydrogen-bond donors have the same orientation (Figure 3-3).
R
R
R
R
R
3.6 residues per
helical turn
R
R
R
R
R
R
R
R
▲ FIGU RE 3 -3 The ␣ helix, a common secondary structure
in proteins. The polypeptide backbone (red) is folded into a spiral
that is held in place by hydrogen bonds between backbone
oxygen and hydrogen atoms. The outer surface of the helix is
covered by the side-chain R groups (green).
62
CHAPTER 3 • Protein Structure and Function
䉴 FIGU RE 3 -4 The  sheet, another common
secondary structure in proteins. (a) Top view of
a simple two-stranded  sheet w ith antiparallel 
strands. The stabilizing hydrogen bonds between
the  strands are indicated by green dashed lines.
The short turn between the  strands also is stabilized
by a hydrogen bond. (b) Side view of a  sheet.
The projection of the R groups (green) above and
below the plane of the sheet is obvious in this view.
The fixed angle of the peptide bond produces a
pleated contour.
(a)
R
R
R
R
R
R
R
R
R
R
R
(b)
R
R
The stable arrangement of amino acids in the ␣ helix holds
the backbone in a rodlike cylinder from which the side chains
point outward. The hydrophobic or hydrophilic quality of the
helix is determined entirely by the side chains because the
polar groups of the peptide backbone are already engaged in
hydrogen bonding in the helix.
The  Sheet Another type of secondary structure, the  sheet,
consists of laterally packed  strands. Each  strand is a short
(5- to 8-residue), nearly fully extended polypeptide segment.
H ydrogen bonding between backbone atoms in adjacent 
strands, within either the same polypeptide chain or between
different polypeptide chains, forms a  sheet (Figure 3-4a). The
planarity of the peptide bond forces a  sheet to be pleated;
hence this structure is also called a  pleated sheet, or simply a
pleated sheet. Like ␣ helices,  strands have a directionality defined by the orientation of the peptide bond. Therefore, in a
pleated sheet, adjacent  strands can be oriented in the same
(parallel) or opposite (antiparallel) directions with respect to
each other. In both arrangements, the side chains project from
both faces of the sheet (Figure 3-4b). In some proteins,  sheets
form the floor of a binding pocket; the hydrophobic core of
other proteins contains multiple  sheets.
Turns Composed of three or four residues, turns are located
on the surface of a protein, forming sharp bends that redirect
the polypeptide backbone back toward the interior. These
short, U-shaped secondary structures are stabilized by a hydrogen bond between their end residues (see Figure 3-4a).
Glycine and proline are commonly present in turns. The lack
of a large side chain in glycine and the presence of a built-in
bend in proline allow the polypeptide backbone to fold into
a tight U shape. Turns allow large proteins to fold into highly
compact structures. A polypeptide backbone also may contain longer bends, or loops. In contrast with turns, which ex-
R
R
R
R
R
R R
R
R
R
R
R
R R
hibit just a few well-defined structures, loops can be formed
in many different ways.
Overall Folding of a Polypeptide Chain Yields
Its Tertiary Structure
Tertiary structure refers to the overall conformation of a
polypeptide chain—that is, the three-dimensional arrangement of all its amino acid residues. In contrast with secondary structures, which are stabilized by hydrogen bonds,
tertiary structure is primarily stabilized by hydrophobic interactions between the nonpolar side chains, hydrogen bonds
between polar side chains, and peptide bonds. These stabilizing forces hold elements of secondary structure—␣ helices,
 strands, turns, and random coils—compactly together.
Because the stabilizing interactions are weak, however, the
tertiary structure of a protein is not rigidly fixed but undergoes continual and minute fluctuation. This variation in
structure has important consequences in the function and
regulation of proteins.
Different ways of depicting the conformation of proteins
convey different types of information. The simplest way to
represent three-dimensional structure is to trace the course of
the backbone atoms with a solid line (Figure 3-5a); the most
complex model shows every atom (Figure 3-5b). The former,
a C ␣ trace, shows the overall organization of the polypeptide
chain without consideration of the amino acid side chains;
the latter, a ball-and-stick model, details the interactions between side-chain atoms, which stabilize the protein’s conformation, as well as the atoms of the backbone. Even though
both views are useful, the elements of secondary structure are
not easily discerned in them. Another type of representation
uses common shorthand symbols for depicting secondary
structure—for example, coiled ribbons or solid cylinders for
␣ helices, flat ribbons or arrows for  strands, and flexible
3.1 • Hierarchical Structure of Proteins
(a) Cα backbone trace
(c) Ribbons
(b) Ball and stick
䉳 FIGU RE 3 -5 Various graphic
(d) Solvent-accessible surface
thin strands for turns and loops (Figure 3-5c). This type of
representation makes the secondary structures of a protein
easy to see.
H owever, none of these three ways of representing protein structure convey much information about the protein
surface, which is of interest because it is where other molecules bind to a protein. Computer analysis can identify the
surface atoms that are in contact with the watery environment. O n this water-accessible surface, regions having a
common chemical character (hydrophobicity or hydrophilicity) and electrical character (basic or acidic) can be mapped.
Such models reveal the topography of the protein surface and
the distribution of charge, both important features of binding sites, as well as clefts in the surface where small molecules often bind (Figure 3-5d). This view represents a protein
as it is “ seen” by another molecule.
Motifs Are Regular Combinations of Secondary
Structures
Particular combinations of secondary structures, called motifs or folds, build up the tertiary structure of a protein. In
some cases, motifs are signatures for a specific function. For
example, the helix-loop-helix is a Ca 2⫹-binding motif
marked by the presence of certain hydrophilic residues at invariant positions in the loop (Figure 3-6a). O xygen atoms in
63
representations of the structure of Ras,
a monomeric guanine nucleotide-binding
protein. The inactive, guanosine
diphosphate (GDP)–bound form is show n
in all four panels, w ith GDP always depicted
in blue spacefill. (a) The C␣ backbone trace
demonstrates how the polypeptide is
packed into the smallest possible volume.
(b) A ball-and-stick representation reveals
the location of all atoms. (c) A ribbon
representation emphasizes how  strands
(blue) and ␣ helices (red) are organized in
the protein. Note the turns and loops
connecting pairs of helices and strands.
(d) A model of the water-accessible surface
reveals the numerous lumps, bumps, and
crevices on the protein surface. Regions of
positive charge are shaded blue; regions of
negative charge are shaded red.
the invariant residues bind a Ca 2⫹ ion through ionic bonds.
This motif, also called the EF hand, has been found in more
than 100 calcium-binding proteins. In another common
motif, the zinc finger, three secondary structures—an ␣ helix
and two  strands with an antiparallel orientation—form a
fingerlike bundle held together by a zinc ion (Figure 3-6b).
This motif is most commonly found in proteins that bind
RN A or DN A.
M any proteins, especially fibrous proteins, self-associate
into oligomers by using a third motif, the coiled coil. In these
proteins, each polypeptide chain contains ␣-helical segments
in which the hydrophobic residues, although apparently
randomly arranged, are in a regular pattern—a repeated
heptad sequence. In the heptad, a hydrophobic residue—
sometimes valine, alanine, or methionine—is at position 1
and a leucine residue is at position 4. Because hydrophilic
side chains extend from one side of the helix and hydrophobic side chains extend from the opposite side, the overall helical structure is amphipathic. The amphipathic character of
these ␣ helices permits two, three, or four helices to wind
around each other, forming a coiled coil; hence the name of
this motif (Figure 3-6c).
We will encounter numerous additional motifs in later
discussions of other proteins in this chapter and other chapters. The presence of the same motif in different proteins
with similar functions clearly indicates that these useful
64
CHAPTER 3 • Protein Structure and Function
(c) Coiled coil m otif
N
(a) Helix-loop-helix m otif
Ca2+
N
(b) Zinc-finger m otif
Leu (4)
Asn
Asp
C
Thr
His
Zn 2+
Val (1)
H2O
Glu
Asp
Cys
N
Leu (4)
His
Asn (1)
Cys
Leu (4)
Val (1)
N
Leu (4)
C
Consensus sequence:
F/Y - C - - C - - - - F/Y - - - - - - - - H - - - H C
Consensus sequence:
D/N - D/N - D/N/S - [backbone O] - - - - E/D
▲ FIGU RE 3 -6 M otifs of protein secondary structure.
(a) Two helices connected by a short loop in a specific
conformation constitute a helix-loop-helix motif. This motif exists
in many calcium-binding and DNA-binding regulatory proteins.
In calcium-binding proteins such as calmodulin, oxygen atoms
from five loop residues and one water molecule form ionic bonds
w ith a Ca2⫹ ion. (b) The zinc-finger motif is present in many
DNA-binding proteins that help regulate transcription. A Zn2⫹ ion
is held between a pair of  strands (blue) and a single ␣ helix
(red) by a pair of cysteine residues and a pair of histidine
residues. The two invariant cysteine residues are usually at
positions 3 and 6 and the two invariant histidine residues are
combinations of secondary structures have been conserved in
evolution. To date, hundreds of motifs have been cataloged
and proteins are now classified according to their motifs.
Structural and Functional Domains Are Modules
of Tertiary Structure
The tertiary structure of proteins larger than 15,000 M W is
typically subdivided into distinct regions called domains.
Structurally, a domain is a compactly folded region of
polypeptide. For large proteins, domains can be recognized
in structures determined by x-ray crystallography or in images captured by electron microscopy. Although these discrete regions are well distinguished or physically separated
from one another, they are connected by intervening segments of the polypeptide chain. Each of the subunits in
hemagglutinin, for example, contains a globular domain and
a fibrous domain (Figure 3-7a).
C
Heptad repeat:
[V/N/M ] - - L - - -
at positions 20 and 24 in this 25-residue motif. (c) The parallel
two-stranded coiled-coil motif found in the transcription factor
Gcn4 is characterized by two ␣ helices wound around one
another. Helix packing is stabilized by interactions between
hydrophobic side chains (red and blue) present at regular
intervals along the surfaces of the intertw ined helices. Each ␣
helix exhibits a characteristic heptad repeat sequence w ith a
hydrophobic residue at positions 1 and 4. [See A. Lew it-Bentley
and S. Rety, 2000, EF-hand calcium-binding proteins, Curr. Opin. Struct.
Biol. 10:637–643; S. A. Wolfe, L. Nekludova, and C. O. Pabo, 2000,
DNA recognition by Cys2His2 zinc finger proteins, Ann. Rev. Biophys.
Biomol. Struct. 29:183–212.]
A structural domain consists of 100–150 residues in various combinations of motifs. O ften a domain is characterized
by some interesting structural feature: an unusual abundance
of a particular amino acid (e.g., a proline-rich domain, an
acidic domain), sequences common to (conserved in) many
proteins (e.g., SH 3, or Src homology region 3), or a particular secondary-structure motif (e.g., zinc-finger motif in the
kringle domain).
Domains are sometimes defined in functional terms on
the basis of observations that an activity of a protein is localized to a small region along its length. For instance, a particular region or regions of a protein may be responsible for
its catalytic activity (e.g., a kinase domain) or binding ability
(e.g., a DN A-binding domain, a membrane-binding domain).
Functional domains are often identified experimentally by
whittling down a protein to its smallest active fragment with
the aid of proteases, enzymes that cleave the polypeptide
backbone. Alternatively, the DN A encoding a protein can be
3.1 • Hierarchical Structure of Proteins
(a)
(b)
Sialic acid
HA2
DISTAL
PROXIM AL
Globular
domain
Fibrous
domain
N
HA1
N
C
Viral
membrane
subjected to mutagenesis so that segments of the protein’s
backbone are removed or changed. The activity of the truncated or altered protein product synthesized from the mutated gene is then monitored and serves as a source of insight
about which part of a protein is critical to its function.
The organization of large proteins into multiple domains illustrates the principle that complex molecules are
built from simpler components. Like motifs of secondary
structure, domains of tertiary structure are incorporated as
modules into different proteins. In Chapter 10 we consider
the mechanism by which the gene segments that correspond
to domains became shuffled in the course of evolution, resulting in their appearance in many proteins. The modular
approach to protein architecture is particularly easy to recognize in large proteins, which tend to be mosaics of different domains and thus can perform different functions
simultaneously.
The epidermal growth factor (EGF) domain is one example of a module that is present in several proteins (Figure 3-8).
EGF is a small, soluble peptide hormone that binds to cells in
the embryo and in skin and connective tissue in adults, causing them to divide. It is generated by proteolytic cleavage between repeated EGF domains in the EGF precursor protein,
which is anchored in the cell membrane by a membranespanning domain. EGF modules are also present in other
proteins and are liberated by proteolysis; these proteins include tissue plasminogen activator (TPA), a protease that is
used to dissolve blood clots in heart attack victims;
65
䉳 FIGU RE 3 -7 Tertiary and quaternary
levels of structure in hemagglutinin (HA),
a surface protein on influenza virus. This
long multimeric molecule has three identical
subunits, each composed of two polypeptide
chains, HA1 and HA2. (a) Tertiary structure of
each HA subunit constitutes the folding of its
helices and strands into a compact structure
that is 13.5 nm long and divided into two
domains. The membrane-distal domain is
folded into a globular conformation. The
membrane-proximal domain has a fibrous,
stemlike conformation ow ing to the alignment
of two long ␣ helices (cylinders) of HA2 w ith
 strands in HA1. Short turns and longer
loops, w hich usually lie at the surface of the
molecule, connect the helices and strands in
a given chain. (b) Quaternary structure of HA
is stabilized by lateral interactions between
the long helices (cylinders) in the fibrous
domains of the three subunits (yellow, blue,
and green), forming a triple-stranded coiledcoil stalk. Each of the distal globular domains
in HA binds sialic acid (red) on the surface of
target cells. Like many membrane proteins,
HA contains several covalently linked
carbohydrate chains (not show n).
N eu protein, which takes part in embryonic differentiation;
and N otch protein, a receptor protein in the plasma membrane that functions in developmentally important signaling
(Chapter 14). Besides the EGF domain, these proteins contain domains found in other proteins. For example, TPA possesses a trypsin domain, a common feature in enzymes that
degrade proteins.
EGF
Neu
EGF
precursor
TPA
▲ FIGU RE 3 -8 Schematic diagrams of various proteins
illustrating their modular nature. Epidermal grow th factor
(EGF) is generated by proteolytic cleavage of a precursor protein
containing multiple EGF domains (green) and a membranespanning domain (blue). The EGF domain is also present in Neu
protein and in tissue plasminogen activator (TPA). These proteins
also contain other w idely distributed domains indicated by shape
and color. [Adapted from I. D. Campbell and P. Bork, 1993, Curr. Opin.
Struct. Biol. 3:385.]
66
CHAPTER 3 • Protein Structure and Function
Proteins Associate into Multimeric Structures
and Macromolecular Assemblies
Multimeric proteins consist of two or more polypeptides or
subunits. A fourth level of structural organization, quaternary
structure, describes the number (stoichiometry) and relative
positions of the subunits in multimeric proteins. H emagglutinin, for example, is a trimer of three identical subunits held
together by noncovalent bonds (Figure 3-7b). O ther multimeric proteins can be composed of any number of identical or
different subunits. The multimeric nature of many proteins
is critical to mechanisms for regulating their function. In addition, enzymes in the same pathway may be associated as
subunits of a large multimeric protein within the cell, thereby
increasing the efficiency of pathway operation.
The highest level of protein structure is the association
of proteins into macromolecular assemblies. Typically, such
structures are very large, exceeding 1 mDa in mass, approaching 30–300 nm in size, and containing tens to hundreds of polypeptide chains, as well as nucleic acids in some
TABLE 3-1
cases. M acromolecular assemblies with a structural function
include the capsid that encases the viral genome and bundles
of cytoskeletal filaments that support and give shape to the
plasma membrane. O ther macromolecular assemblies act as
molecular machines, carrying out the most complex cellular
processes by integrating individual functions into one coordinated process. For example, the transcriptional machine
that initiates the synthesis of messenger RN A (mRN A) consists of RN A polymerase, itself a multimeric protein, and at
least 50 additional components including general transcription factors, promoter-binding proteins, helicase, and other
protein complexes (Figure 3-9). The transcription factors
and promoter-binding proteins correctly position a polymerase molecule at a promoter, the DN A site that determines
where transcription of a specific gene begins. After helicase
unwinds the double-stranded DN A molecule, polymerase simultaneously translocates along the DN A template strand
and synthesizes an mRN A strand. The operational details of
this complex machine and of others listed in Table 3-1 are
discussed elsewhere.
Selected Molecular Machines
Machine*
Main Components
Cellular Location
Function
Replisome (4)
H elicase, primase, DN A polymerase
N ucleus
DN A replication
Transcription initiation
complex (11)
Promoter-binding protein, helicase,
general transcription factors (TFs), RN A
polymerase, large multisubunit mediator
complex
N ucleus
RN A synthesis
Spliceosome (12)
Pre-mRN A, small nuclear RN As
(snRN As), protein factors
N ucleus
mRN A splicing
N uclear pore
complex (12)
N ucleoporins (50–100)
N uclear membrane
N uclear import
and export
Ribosome (4)
Ribosomal proteins (⬎50) and four
rRN A molecules (eukaryotes) organized
into large and small subunits; associated
mRN A and protein factors (IFs, EFs)
Cytoplasm/ER membrane
Protein synthesis
Chaperonin (3)
GroEL, GroES (bacteria)
Cytoplasm,
mitochondria,
endoplasmic
reticulum
Protein folding
Proteasome (3)
Core proteins, regulatory (cap) proteins
Cytoplasm
Protein degradation
Photosystem (8)
Light-harvesting complex (multiple
proteins and pigments), reaction center
(multisubunit protein with associated
pigments and electron carriers)
Thylakoid membrane
in plant chloroplasts,
plasma membrane of
photosynthetic bacteria
Photosynthesis
(initial stage)
M AP kinase
cascades (14)
Scaffold protein, multiple different
protein kinases
Cytoplasm
Signal transduction
Sarcomere (19)
Thick (myosin) filaments, thin (actin)
filaments, Z lines, titin/nebulin
Cytoplasm of
muscle cells
Contraction
*
N umbers in parentheses indicate chapters in which various machines are discussed.
3.1 • Hierarchical Structure of Proteins
Members of Protein Families Have a Common
Evolutionary Ancestor
General transcription factors
+
67
+
RNA polym erase
M ediator
com plex
DNA
Prom oter
Transcription
preinitiation
com plex
▲ FIGU RE 3 -9 The mRNA transcription-initiation machinery.
The core RNA polymerase, general transcription factors, a
mediator complex containing about 20 subunits, and other
protein complexes not depicted here assemble at a promoter in
DNA. The polymerase carries out transcription of DNA; the
associated proteins are required for initial binding of polymerase
to a specific promoter, thereby initiating transcription.
Studies on myoglobin and hemoglobin, the oxygen-carrying
proteins in muscle and blood, respectively, provided early evidence that function derives from three-dimensional structure, which in turn is specified by amino acid sequence.
X-ray crystallographic analysis showed that the threedimensional structures of myoglobin and the ␣ and  subunits of hemoglobin are remarkably similar. Subsequent sequencing of myoglobin and the hemoglobin subunits
revealed that many identical or chemically similar residues
are found in identical positions throughout the primary
structures of both proteins.
Similar comparisons between other proteins conclusively
confirmed the relation between the amino acid sequence,
three-dimensional structure, and function of proteins. This
principle is now commonly employed to predict, on the
basis of sequence comparisons with proteins of known
structure and function, the structure and function of proteins that have not been isolated (Chapter 9). This use of
sequence comparisons has expanded substantially in recent
years as the genomes of more and more organisms have
been sequenced.
The molecular revolution in biology during the last
decades of the twentieth century also created a new scheme
α
α
Vertebrate
HEM OGLOBIN
α
β
M YOGLOBIN
Dicot
hem oglobin
Annelid
LEGHEM OGLOBIN
M onocot
hem oglobin
Insect
Nem atode
β
β
Hem oglobin
Protozoan
Algal
Fungal
Bacterial
Ancestral
oxygen-binding
protein
▲ FIGU RE 3 -10 Evolution of the globin protein family. (Left)
A primitive monomeric oxygen-binding globin is thought to be the
ancestor of modern-day blood hemoglobins, muscle myoglobins,
and plant leghemoglobins. Sequence comparisons have revealed
that evolution of the globin proteins parallels the evolution of
animals and plants. M ajor junctions occurred w ith the divergence
of plant globins from animal globins and of myoglobin from
hemoglobin. Later gene duplication gave rise to the ␣ and 
Leghem oglobin
β subunit
of hem oglobin
M yoglobin
subunits of hemoglobin. (Right) Hemoglobin is a tetramer of two
␣ and two  subunits. The structural similarity of these subunits
w ith leghemoglobin and myoglobin, both of w hich are
monomers, is evident. A heme molecule (red) noncovalently
associated w ith each globin polypeptide is the actual oxygenbinding moiety in these proteins. [(Left) Adapted from R. C.
Hardison, 1996, Proc. Natl. Acad. Sci. USA 93:5675.]
68
CHAPTER 3 • Protein Structure and Function
of biological classification based on similarities and differences in the amino acid sequences of proteins. Proteins that
have a common ancestor are referred to as hom ologs. The
main evidence for homology among proteins, and hence
their common ancestry, is similarity in their sequences or
structures. We can therefore describe homologous proteins
as belonging to a “ family” and can trace their lineage from
comparisons of their sequences. The folded three-dimensional structures of homologous proteins are similar even if
parts of their primary structure show little evidence of
homology.
The kinship among homologous proteins is most easily
visualized by a tree diagram based on sequence analyses. For
example, the amino acid sequences of globins from bacteria,
plants, and animals suggest that they evolved from an ancestral monomeric, oxygen-binding protein (Figure 3-10).
With the passage of time, the gene for this ancestral protein
slowly changed, initially diverging into lineages leading to
animal and plant globins. Subsequent changes gave rise to
myoglobin, a monomeric oxygen-storing protein in muscle,
and to the ␣ and  subunits of the tetrameric hemoglobin
molecule (␣2 2 ) of the circulatory system.
K EY C O N C EP T S O F S EC T I O N 3 . 1
Hierarchical Structure of Proteins
■ A protein is a linear polymer of amino acids linked
together by peptide bonds. Various, mostly noncovalent,
interactions between amino acids in the linear sequence
stabilize a specific folded three-dimensional structure (conformation) for each protein.
The ␣ helix,  strand and sheet, and turn are the most
prevalent elements of protein secondary structure, which
is stabilized by hydrogen bonds between atoms of the peptide backbone.
■
■ Certain combinations of secondary structures give rise
to different motifs, which are found in a variety of proteins and are often associated with specific functions (see
Figure 3-6).
■ Protein tertiary structure results from hydrophobic interactions between nonpolar side groups and hydrogen
bonds between polar side groups that stabilize folding of
the secondary structure into a compact overall arrangement, or conformation.
Large proteins often contain distinct domains, independently folded regions of tertiary structure with characteristic
structural or functional properties or both (see Figure 3-7).
■
■ The incorporation of domains as modules in different
proteins in the course of evolution has generated diversity
in protein structure and function.
■ Q uaternary structure encompasses the number and organization of subunits in multimeric proteins.
Cells contain large macromolecular assemblies in which
all the necessary participants in complex cellular processes
(e.g., DN A, RN A, and protein synthesis; photosynthesis;
signal transduction) are integrated to form molecular machines (see Table 3-1).
■
■ The sequence of a protein determines its three-dimensional
structure, which determines its function. In short, function
derives from structure; structure derives from sequence.
H omologous proteins, which have similar sequences,
structures, and functions, evolved from a common ancestor.
■
3.2 Folding, Modification,
and Degradation of Proteins
A polypeptide chain is synthesized by a complex process called
translation in which the assembly of amino acids in a particular sequence is dictated by messenger RN A (mRN A). The intricacies of translation are considered in Chapter 4. H ere, we
describe how the cell promotes the proper folding of a nascent polypeptide chain and, in many cases, modifies residues
or cleaves the polypeptide backbone to generate the final protein. In addition, the cell has error-checking processes that
eliminate incorrectly synthesized or folded proteins. Incorrectly folded proteins usually lack biological activity and, in
some cases, may actually be associated with disease. Protein
misfolding is suppressed by two distinct mechanisms. First,
cells have systems that reduce the chances for misfolded proteins to form. Second, any misfolded proteins that do form,
as well as cytosolic proteins no longer needed by a cell, are degraded by a specialized cellular garbage-disposal system.
The Information for Protein Folding Is Encoded
in the Sequence
Any polypeptide chain containing n residues could, in principle, fold into 8 n conformations. This value is based on the
fact that only eight bond angles are stereochemically allowed
in the polypeptide backbone. In general, however, all molecules of any protein species adopt a single conformation,
called the native state; for the vast majority of proteins, the
native state is the most stably folded form of the molecule.
What guides proteins to their native folded state? The answer to this question initially came from in vitro studies on
protein refolding. Thermal energy from heat, extremes of pH
that alter the charges on amino acid side chains, and chemicals such as urea or guanidine hydrochloride at concentrations of 6–8 M can disrupt the weak noncovalent interactions
that stabilize the native conformation of a protein. The
denaturation resulting from such treatment causes a protein
to lose both its native conformation and its biological activity.
M any proteins that are completely unfolded in 8 M urea
and -mercaptoethanol (which reduces disulfide bonds) spontaneously renature (refold) into their native states when the denaturing reagents are removed by dialysis. Because no cofactors
3.2 • Folding, Modification, and Degradation of Proteins
69
or other proteins are required, in vitro protein folding is a selfdirected process. In other words, sufficient information must
be contained in the protein’s primary sequence to direct correct refolding. The observed similarity in the folded, threedimensional structures of proteins with similar amino acid
sequences, noted in Section 3.1, provided other evidence that
the primary sequence also determines protein folding in vivo.
class of proteins found in all organisms from bacteria to humans. Chaperones are located in every cellular compartment,
bind a wide range of proteins, and function in the general
protein-folding mechanism of cells. Two general families of
chaperones are reconized:
Folding of Proteins in Vivo Is Promoted
by Chaperones
Chaperonins, which directly facilitate the folding of
proteins
Although protein folding occurs in vitro, only a minority of
unfolded molecules undergo complete folding into the native
conformation within a few minutes. Clearly, cells require a
faster, more efficient mechanism for folding proteins into
their correct shapes; otherwise, cells would waste much energy in the synthesis of nonfunctional proteins and in the
degradation of misfolded or unfolded proteins. Indeed, more
than 95 percent of the proteins present within cells have been
shown to be in their native conformation, despite high protein concentrations (200–300 mg/ml), which favor the precipitation of proteins in vitro.
The explanation for the cell’s remarkable efficiency in
promoting protein folding probably lies in chaperones, a
Molecular chaperones consist of H sp70 and its homologs:
H sp70 in the cytosol and mitochondrial matrix, BiP in the endoplasmic reticulum, and DnaK in bacteria. First identified
by their rapid appearance after a cell has been stressed by heat
shock, H sp70 and its homologs are the major chaperones in
all organisms. (H sc70 is a constitutively expressed homolog of
H sp70.) When bound to ATP, H sp70-like proteins assume an
open form in which an exposed hydrophobic pocket transiently binds to exposed hydrophobic regions of the unfolded
target protein. H ydrolysis of the bound ATP causes molecular chaperones to assume a closed form in which a target protein can undergo folding. The exchange of ATP for ADP
releases the target protein (Figure 3-11a, top). This cycle is
■ M olecular chaperones, which bind and stabilize unfolded or partly folded proteins, thereby preventing these
proteins from aggregating and being degraded
■
(a)
(b)
Ribosom e
Protein
Partially
folded
protein
ATP
Properly
folded
protein
GroEL "tight "
conform ation
ADP
+
Pi
Protein
Properly
folded
protein
ATP
GroES
GroEL
▲ FIGU RE 3 -11 Chaperone- and chaperonin-mediated
protein folding. (a) M any proteins fold into their proper threedimensional structures w ith the assistance of Hsp70-like proteins
(top). These molecular chaperones transiently bind to a nascent
polypeptide as it emerges from a ribosome. Proper folding of
other proteins (bottom) depends on chaperonins such as the
prokaryotic GroEL, a hollow, barrel-shaped complex of 14
identical 60,000-M W subunits arranged in two stacked rings.
GroEL "relaxed"
conform ation
One end of GroEL is transiently blocked by the cochaperonin GroES, an assembly of 10,000-M W subunits.
(b) In the absence of ATP or presence of ADP, GroEL exists
in a “ tight” conformational state that binds partly folded or
misfolded proteins. Binding of ATP shifts GroEL to a more
open, “ relaxed” state, w hich releases the folded protein.
See text for details. [Part (b) from A. Roseman et al., 1996, Cell
87:241; courtesy of H. Saibil.]
M EDIA CON N ECTION S
Hsp 70-ATP
ADP
Focus Animation: Chaperone-Mediated Folding
Pi
70
CHAPTER 3 • Protein Structure and Function
speeded by the co-chaperone Hsp40 in eukaryotes. In bacteria,
an additional protein called GrpE also interacts with DnaK,
promoting the exchange of ATP for the bacterial co-chaperone
DnaJ and possibly its dissociation. M olecular chaperones are
thought to bind all nascent polypeptide chains as they are
being synthesized on ribosomes. In bacteria, 85 percent of the
proteins are released from their chaperones and proceed to
fold normally; an even higher percentage of proteins in eukaryotes follow this pathway.
The proper folding of a large variety of newly synthesized
or translocated proteins also requires the assistance of chaperonins. These huge cylindrical macromolecular assemblies are
formed from two rings of oligomers. The eukaryotic chaperonin TriC consists of eight subunits per ring. In the bacterial,
mitochondrial, and chloroplast chaperonin, known as GroEL,
each ring contains seven identical subunits (Figure 3-11b). The
GroEL folding mechanism, which is better understood than
TriC-mediated folding, serves as a general model (Figure
3-11a, bottom ). In bacteria, a partly folded or misfolded
polypeptide is inserted into the cavity of GroEL, where it binds
to the inner wall and folds into its native conformation. In an
ATP-dependent step, GroEL undergoes a conformational
change and releases the folded protein, a process assisted by a
co-chaperonin, GroES, which caps the ends of GroEL.
Many Proteins Undergo Chemical Modification
of Amino Acid Residues
N early every protein in a cell is chemically modified after its
synthesis on a ribosome. Such modifications, which may
alter the activity, life span, or cellular location of proteins,
entail the linkage of a chemical group to the free –N H 2 or
–CO O H group at either end of a protein or to a reactive sidechain group in an internal residue. Although cells use the 20
amino acids shown in Figure 2-13 to synthesize proteins,
analysis of cellular proteins reveals that they contain upward
of 100 different amino acids. Chemical modifications after
synthesis account for this difference.
A cetylation, the addition of an acetyl group (CH 3 CO ) to
the amino group of the N -terminal residue, is the most common form of chemical modification, affecting an estimated
80 percent of all proteins:
R
O
N
C
C
H
H
O
CH3
C
O
Acetyl lysine
CH3
C
N
CH2
CH2
CH2
CH
CH2
COOⴚ
NH3ⴙ
O
Phosphoserine
−O
P
CH2
O
CH
COOⴚ
NH3ⴙ
O−
OH
3-Hydroxyproline
H2C
CH
H2C
CH
COOⴚ
ⴙ
NH2
HC
3-M ethylhistidine
H3C
ⴚ
N
C
C
H
CH2
CH
COOⴚ
NH3ⴙ
N
OOC
␥ -Carboxyglutamate
CH
CH2
CH
COOⴚ
ⴚ
OOC
NH3ⴙ
▲ FIGU RE 3 -12 Common modifications of internal amino
acid residues found in proteins. These modified residues and
numerous others are formed by addition of various chemical
groups (red) to the amino acid side chains after synthesis of a
polypeptide chain.
Acetyl groups and a variety of other chemical groups can
also be added to specific internal residues in proteins (Figure 3-12). An important modification is the phosphorylation
of serine, threonine, tyrosine, and histidine residues. We will
encounter numerous examples of proteins whose activity is
regulated by reversible phosphorylation and dephosphorylation. The side chains of asparagine, serine, and threonine
are sites for glycosylation, the attachment of linear and
branched carbohydrate chains. M any secreted proteins and
membrane proteins contain glycosylated residues; the synthesis of such proteins is described in Chapters 16 and 17.
O ther post-translational modifications found in selected proteins include the hydrox ylation of proline and lysine residues
in collagen, the m ethylation of histidine residues in membrane receptors, and the ␥-carbox ylation of glutamate in
prothrombin, an essential blood-clotting factor. A special
modification, discussed shortly, marks cytosolic proteins for
degradation.
Acetylated N-terminus
This modification may play an important role in controlling
the life span of proteins within cells because nonacetylated
proteins are rapidly degraded by intracellular proteases.
Residues at or near the termini of some membrane proteins are
chemically modified by the addition of long lipidlike groups.
The attachment of these hydrophobic “ tails,” which function
to anchor proteins to the lipid bilayer, constitutes one way that
cells localize certain proteins to membranes (Chapter 5).
Peptide Segments of Some Proteins Are Removed
After Synthesis
After their synthesis, some proteins undergo irreversible
changes that do not entail changes in individual amino acid
residues. This type of post-translational alteration is sometimes called processing. The most common form is enzymatic
cleavage of a backbone peptide bond by proteases, resulting
in the removal of residues from the C- or N -terminus of a
71
3.2 • Folding, Modification, and Degradation of Proteins
(a)
NH2
Ub
AM P
+ PPi
+ ATP
C
O E2
E1
Ub
E1
1
C
Cytosolic
target protein
O
Ub
2
3
E3
E2
O
E1 = Ubiquitin-activating enzym e
NH
E2 = Ubiquitin-conjugating enzym e
C
Ub
E3 = Ubiquitin ligase
Ub = Ubiquitin
Steps 1, 2, 3
(n tim es)
(b)
Ub
Ub
Ub
n
Cap
ATP
4
ADP
Core
Ubiquitin Marks Cytosolic Proteins
for Degradation in Proteasomes
In addition to chemical modifications and processing, the activity of a cellular protein depends on the amount present,
which reflects the balance between its rate of synthesis and
rate of degradation in the cell. The numerous ways that cells
regulate protein synthesis are discussed in later chapters. In
this section, we examine protein degradation, focusing on
the major pathways for degrading cytosolic proteins.
The life span of intracellular proteins varies from as short
as a few minutes for mitotic cyclins, which help regulate passage through mitosis, to as long as the age of an organism for
proteins in the lens of the eye. Eukaryotic cells have several
intracellular proteolytic pathways for degrading misfolded or
denatured proteins, normal proteins whose concentration
must be decreased, and extracellular proteins taken up by the
cell. O ne major intracellular pathway is degradation by enzymes within lysosomes, membrane-limited organelles whose
acidic interior is filled with hydrolytic enzymes. Lysosomal
degradation is directed primarily toward extracellular proteins taken up by the cell and aged or defective organelles of
the cell (see Figure 5-20).
Distinct from the lysosomal pathway are cytosolic mechanisms for degrading proteins. Chief among these mechanisms
is a pathway that includes the chemical modification of a lysine side chain by the addition of ubiquitin, a 76-residue
polypeptide, followed by degradation of the ubiquitin-tagged
protein by a specialized proteolytic machine. Ubiquitination
is a three-step process (Figure 3-13a):
■ Activation of ubiquitin-activating enzym e (E1) by the
addition of a ubitiquin molecule, a reaction that requires
ATP
■ Transfer of this ubiquitin molecule to a cysteine residue
in ubiquitin-conjugating enzym e (E2)
Proteasom e
Ub
Cap
Ub
5
Ub
Peptides
▲ FIGU RE 3 -13 Ubiquitin-mediated proteolytic
pathway. (a) Enzyme E1 is activated by attachment of a
ubiquitin (Ub) molecule (step 1 ) and then transfers this Ub
molecule to E2 (step 2 ). Ubiquitin ligase (E3) transfers the
bound Ub molecule on E2 to the side-chain —NH2 of a lysine
residue in a target protein (step 3 ). Additional Ub molecules
are added to the target protein by repeating steps 1 – 3 ,
forming a polyubiquitin chain that directs the tagged protein
to a proteasome (step 4 ). Within this large complex, the
protein is cleaved into numerous small peptide fragments
(step 5 ). (b) Computer-generated image reveals that a
proteasome has a cylindrical structure w ith a cap at each end
of a core region. Proteolysis of ubiquitin-tagged proteins
occurs along the inner wall of the core. [Part (b) from
W. Baumeister et al., 1998, Cell 92:357; courtesy of W. Baumeister.]
Formation of a peptide bond between the ubiquitin
molecule bound to E2 and a lysine residue in the target
protein, a reaction catalyzed by ubiquitin ligase (E3)
■
This process is repeated many times, with each subsequent
ubiquitin molecule being added to the preceding one. The resulting polyubiquitin chain is recognized by a proteasome,
another of the cell’s molecular machines (Figure 3-13b). The
numerous proteasomes dispersed throughout the cell cytosol
proteolytically cleave ubiquitin-tagged proteins in an ATPdependent process that yields short (7- to 8-residue) peptides
and intact ubiquitin molecules.
M EDIA CON N ECTION S
E2
E1
Overview Animation: Life Cycle of a Protein
polypeptide chain. Proteolytic cleavage is a common mechanism for activating enzymes that function in blood coagulation, digestion, and programmed cell death (Chapter 22).
Proteolysis also generates active peptide hormones, such as
EGF and insulin, from larger precursor polypeptides.
An unusual and rare type of processing, termed protein
self-splicing, takes place in bacteria and some eukaryotes.
This process is analogous to editing film: an internal segment
of a polypeptide is removed and the ends of the polypeptide
are rejoined. Unlike proteolytic processing, protein selfsplicing is an autocatalytic process, which proceeds by itself
without the participation of enzymes. The excised peptide
appears to eliminate itself from the protein by a mechanism
similar to that used in the processing of some RN A molecules (Chapter 12). In vertebrate cells, the processing of some
proteins includes self-cleavage, but the subsequent ligation
step is absent. O ne such protein is H edgehog, a membranebound signaling molecule that is critical to a number of developmental processes (Chapter 15).
72
CHAPTER 3 • Protein Structure and Function
Cellular proteins degraded by the ubiquitin-mediated
pathway fall into one of two general categories: (1) native cytosolic proteins whose life spans are tightly controlled and
(2) proteins that become misfolded in the course of their synthesis in the endoplasmic reticulum (ER). Both contain sequences recognized by the ubiquitinating enzyme complex.
The cyclins, for example, are cytosolic proteins whose
amounts are tightly controlled throughout the cell cycle.
These proteins contain the internal sequence Arg-X-X-LeuGly-X-Ile-Gly-Asp/Asn (X can be any amino acid), which is
recognized by specific ubiquitinating enzyme complexes. At
a specific time in the cell cycle, each cyclin is phosphorylated
by a cyclin kinase. This phosphorylation is thought to cause
a conformational change that exposes the recognition sequence to the ubiquitinating enzymes, leading to degradation
of the tagged cyclin (Chapter 21). Similarly, the misfolding of
proteins in the endoplasmic reticulum exposes hydrophobic
sequences normally buried within the folded protein. Such
proteins are transported to the cytosol, where ubiquitinating enzymes recognize the exposed hydrophobic sequences.
The immune system also makes use of the ubiquitinmediated pathway in the response to altered self-cells, particularly virus-infected cells. Viral proteins within the cytosol
of infected cells are ubiquitinated and then degraded in proteasomes specially designed for this role. The resulting antigenic peptides are transported to the endoplasmic reticulum,
where they bind to class I major histocompatibility complex
(M H C) molecules within the ER membrane. Subsequently,
the peptide-M H C complexes move to the cell membrane
where the antigenic peptides can be recognized by cytotoxic
T lymphocytes, which mediate the destruction of the infected
cells.
Alternatively Folded Proteins Are Implicated in
Slowly Developing Diseases
As noted earlier, each protein species normally folds
into a single, energetically favorable conformation
that is specified by its amino acid sequence. Recent
evidence suggests, however, that a protein may fold into an alternative three-dimensional structure as the result of mutations, inappropriate post-translational modification, or other
as-yet-unidentified reasons. Such “ misfolding” not only leads
to a loss of the normal function of the protein but also marks
it for proteolytic degradation. The subsequent accumulation
of proteolytic fragments contributes to certain degenerative
diseases characterized by the presence of insoluble protein
plaques in various organs, including the liver and brain. ❚
Some neurodegenerative diseases, including Alzheimer’s
disease and Parkinson’s disease in humans and transmissible
spongiform encephalopathy (“ mad cow” disease) in cows
(b)
(a)
Digestive Proteases Degrade Dietary Proteins
The major extracellular pathway for protein degradation is the
system of digestive proteases that breaks down ingested proteins into peptides and amino acids in the intestinal tract.
Three classes of proteases function in digestion. Endoproteases
attack selected peptide bonds within a polypeptide chain. The
principal endoproteases are pepsin, which preferentially
cleaves the backbone adjacent to phenylalanine and leucine
residues, and trypsin and chymotrypsin, which cleave the
backbone adjacent to basic and aromatic residues. Ex opeptidases sequentially remove residues from the N -terminus
(aminopeptidases) or C-terminus (carboxypeptidases) of a
protein. Peptidases split oligopeptides containing as many as
about 20 amino acids into di- and tripeptides and individual
amino acids. These small molecules are then transported
across the intestinal lining into the bloodstream.
To protect a cell from degrading itself, endoproteases and
carboxypeptidases are synthesized and secreted as inactive
forms (zymogens): pepsin by chief cells in the lining of the
stomach; the others by pancreatic cells. Proteolytic cleavage
of the zymogens within the gastic or intestinal lumen yields
the active enzymes. Intestinal epithelial cells produce
aminopeptidases and the di- and tripeptidases.
20 m
100 nm
▲ EX PERIM EN TA L FIGU RE 3 -14 Alzheimer’s disease is
characterized by the formation of insoluble plaques
composed of amyloid protein. (a) At low resolution, an amyloid
plaque in the brain of an Alzheimer’s patient appears as a tangle
of filaments. (b) The regular structure of filaments from plaques
is revealed in the atomic force microscope. Proteolysis of the
naturally occurring amyloid precursor protein yields a short
fragment, called -amyloid protein, that for unknow n reasons
changes from an ␣-helical to a -sheet conformation. This
alternative structure aggregates into the highly stable filaments
(amyloid) found in plaques. Similar pathologic changes in other
proteins cause other degenerative diseases. [Courtesy of K. Kosik.]
3.3 • Enzymes and the Chemical Work of Cells
and sheep, are marked by the formation of tangled filamentous plaques in a deteriorating brain (Figure 3-14). The am yloid filam ents composing these structures derive from
abundant natural proteins such as amyloid precursor protein, which is embedded in the plasma membrane, Tau, a
microtubule-binding protein, and prion protein, an “ infectious” protein whose inheritance follows M endelian genetics.
Influenced by unknown causes, these ␣ helix–containing proteins or their proteolytic fragments fold into alternative 
sheet–containing structures that polymerize into very stable
filaments. Whether the extracellular deposits of these filaments or the soluble alternatively folded proteins are toxic to
the cell is unclear.
73
degree of specificity. For instance, an enzyme must first bind
specifically to its target molecule, which may be a small molecule (e.g., glucose) or a macromolecule, before it can execute
its specific task. Likewise, the many different types of hormone receptors on the surface of cells display a high degree of
sensitivity and discrimination for their ligands. And, as we
will examine in Chapter 11, the binding of certain regulatory
proteins to specific sequences in DN A is a major mechanism
for controlling genes. Ligand binding often causes a change in
the shape of a protein. Ligand-driven conformational changes
are integral to the mechanism of action of many proteins and
are important in regulating protein activity. After considering the general properties of protein–ligand binding, we take
a closer look at how enzymes are designed to function as the
cell’s chemists.
K EY C O N C EP T S O F S EC T I O N 3 . 2
Folding, Modification, and Degradation of Proteins
■ The amino acid sequence of a protein dictates its folding into a specific three-dimensional conformation, the native state.
■ Protein folding in vivo occurs with assistance from molecular chaperones (H sp70 proteins), which bind to nascent polypeptides emerging from ribosomes and prevent
their misfolding (see Figure 3-11). Chaperonins, large complexes of H sp60-like proteins, shelter some partly folded
or misfolded proteins in a barrel-like cavity, providing additional time for proper folding.
■ Subsequent to their synthesis, most proteins are modified by the addition of various chemical groups to amino
acid residues. These modifications, which alter protein
structure and function, include acetylation, hydroxylation,
glycosylation, and phosphorylation.
■ The life span of intracellular proteins is largely determined by their susceptibility to proteolytic degradation by
various pathways.
■ Viral proteins produced within infected cells, normal cytosolic proteins, and misfolded proteins are marked for destruction by the covalent addition of a polyubiquitin chain
and then degraded within proteasomes, large cylindrical
complexes with multiple proteases in their interiors (see
Figure 3-13).
■ Some neurodegenerative diseases are caused by aggregates of proteins that are stably folded in an alternative
conformation.
3.3 Enzymes and the Chemical Work
of Cells
Proteins are designed to bind every conceivable molecule—
from simple ions and small metabolites (sugars, fatty acids) to
large complex molecules such as other proteins and nucleic
acids. Indeed, the function of nearly all proteins depends on
their ability to bind other molecules, or ligands, with a high
Specificity and Affinity of Protein–Ligand Binding
Depend on Molecular Complementarity
Two properties of a protein characterize its interaction with
ligands. Specificity refers to the ability of a protein to bind
one molecule in preference to other molecules. A ffinity
refers to the strength of binding. The K d for a protein–
ligand complex, which is the inverse of the equilibrium constant K eq for the binding reaction, is the most common
quantitative measure of affinity (Chapter 2). The stronger
the interaction between a protein and ligand, the lower the
value of K d . Both the specificity and the affinity of a protein
for a ligand depend on the structure of the ligand-binding
site, which is designed to fit its partner like a mold. For
high-affinity and highly specific interactions to take place,
the shape and chemical surface of the binding site must be
complementary to the ligand molecule, a property termed
molecular complementarity.
The ability of proteins to distinguish different molecules
is perhaps most highly developed in the blood proteins called
antibodies, which animals produce in response to antigens,
such as infectious agents (e.g., a bacterium or a virus), and
certain foreign substances (e.g., proteins or polysaccharides
in pollens). The presence of an antigen causes an organism to
make a large quantity of different antibody proteins, each
of which may bind to a slightly different region, or epitope,
of the antigen. Antibodies act as specific sensors for antigens,
forming antibody–antigen complexes that initiate a cascade
of protective reactions in cells of the immune system.
All antibodies are Y-shaped molecules formed from
two identical heavy chains and two identical light chains
(Figure 3-15a). Each arm of an antibody molecule contains
a single light chain linked to a heavy chain by a disulfide
bond. N ear the end of each arm are six highly variable loops,
called com plem entarity-determ ining regions (CD R s), which
form the antigen-binding sites. The sequences of the six loops
are highly variable among antibodies, making them specific
for different antigens. The interaction between an antibody
and an epitope in an antigen is complementary in all cases;
that is, the surface of the antibody’s antigen-binding site
physically matches the corresponding epitope like a glove
74
CHAPTER 3 • Protein Structure and Function
▲ FIGU RE 3 -15 Antibody structure and antibody-antigen
interaction. (a) Ribbon model of an antibody. Every antibody
molecule consists of two identical heavy chains (red) and two
identical light chains (blue) covalently linked by disulfide bonds.
(b) The hand-in-glove fit between an antibody and an epitope on
its antigen—in this case, chicken egg-w hite lysozyme. Regions
(Figure 3-15b). The intimate contact between these two surfaces, stabilized by numerous noncovalent bonds, is responsible for the exquisite binding specificity exhibited by an
antibody.
The specificity of antibodies is so precise that they can
distinguish between the cells of individual members of a
species and in some cases can distinguish between proteins
that differ by only a single amino acid. Because of their specificity and the ease with which they can be produced, antibodies are highly useful reagents in many of the experiments
discussed in subsequent chapters.
Enzymes Are Highly Efficient and Specific
Catalysts
In contrast with antibodies, which bind and simply present
their ligands to other components of the immune system, enzymes promote the chemical alteration of their ligands,
called substrates. Almost every chemical reaction in the cell
is catalyzed by a specific enzyme. Like all catalysts, enzymes
do not affect the extent of a reaction, which is determined by
the change in free energy ⌬G between reactants and products
(Chapter 2). For reactions that are energetically favorable
(⫺⌬G ), enzymes increase the reaction rate by lowering the
activation energy (Figure 3-16). In the test tube, catalysts
such as charcoal and platinum facilitate reactions but usually
only at high temperatures or pressures, at extremes of high
w here the two molecules make contact are show n as surfaces.
The antibody contacts the antigen w ith residues from all its
complementarity-determining regions (CDRs). In this view, the
complementarity of the antigen and antibody is especially
apparent w here “ fingers” extending from the antigen surface are
opposed to “ clefts” in the antibody surface.
or low pH , or in organic solvents. As the cell’s protein catalysts, however, enzymes must function effectively in aqueous
environment at 37⬚C, 1 atmosphere pressure, and pH
6.5–7.5.
Two striking properties of enzymes enable them to function as catalysts under the mild conditions present in cells:
their enormous catalytic pow er and their high degree of
specificity. The immense catalytic power of enzymes causes
the rates of enzymatically catalyzed reactions to be 10 6 –10 12
times that of the corresponding uncatalyzed reactions under
otherwise similar conditions. The exquisite specificity of
enzymes—their ability to act selectively on one substrate or a
small number of chemically similar substrates —is exemplified by the enzymes that act on amino acids. As noted in
Chapter 2, amino acids can exist as two stereoisomers, designated L and D , although only L isomers are normally found
in biological systems. N ot surprisingly, enzyme-catalyzed reactions of L-amino acids take place much more rapidly than
do those of D -amino acids, even though both stereoisomers
of a given amino acid are the same size and possess the same
R groups (see Figure 2-12).
Approximately 3700 different types of enzymes, each of
which catalyzes a single chemical reaction or set of closely related reactions, have been classified in the enzyme database.
Certain enzymes are found in the majority of cells because
they catalyze the synthesis of common cellular products (e.g.,
proteins, nucleic acids, and phospholipids) or take part in the
3.3 • Enzymes and the Chemical Work of Cells
Transition state
(uncatalyzed)
Free energy, G
ΔGⴝuncat
Transition state
(catalyzed)
ΔGⴝcat
Reactants
Products
Progress of reaction
▲ FIGU RE 3 -16 Effect of a catalyst on the activation energy
of a chemical reaction. This hypothetical reaction pathway
depicts the changes in free energy G as a reaction proceeds. A
reaction w ill take place spontaneously only if the total G of the
products is less than that of the reactants (⫺⌬G). However, all
chemical reactions proceed through one or more high-energy
transition states, and the rate of a reaction is inversely
proportional to the activation energy (⌬G‡), w hich is the
difference in free energy between the reactants and the highest
point along the pathway. Enzymes and other catalysts accelerate
the rate of a reaction by reducing the free energy of the
transition state and thus ⌬G‡.
production of energy by the conversion of glucose and oxygen into carbon dioxide and water. O ther enzymes are present only in a particular type of cell because they catalyze
chemical reactions unique to that cell type (e.g., the enzymes
that convert tyrosine into dopamine, a neurotransmitter, in
nerve cells). Although most enzymes are located within cells,
some are secreted and function in extracellular sites such as
the blood, the lumen of the digestive tract, or even outside
the organism.
The catalytic activity of some enzymes is critical to cellular processes other than the synthesis or degradation of molecules. For instance, many regulatory proteins and intracellular
signaling proteins catalyze the phosphorylation of proteins,
and some transport proteins catalyze the hydrolysis of ATP
coupled to the movement of molecules across membranes.
An Enzyme’s Active Site Binds Substrates
and Carries Out Catalysis
Certain amino acid side chains of an enzyme are important
in determining its specificity and catalytic power. In the native conformation of an enzyme, these side chains are
brought into proximity, forming the active site. Active sites
thus consist of two functionally important regions: one that
recognizes and binds the substrate (or substrates) and another that catalyzes the reaction after the substrate has been
75
bound. In some enzymes, the catalytic region is part of the
substrate-binding region; in others, the two regions are structurally as well as functionally distinct.
To illustrate how the active site binds a specific substrate
and then promotes a chemical change in the bound substrate,
we examine the action of cyclic AM P–dependent protein kinase, now generally referred to as protein kinase A (PKA).
This enzyme and other protein kinases, which add a phosphate group to serine, threonine, or tyrosine residues in proteins, are critical for regulating the activity of many cellular
proteins, often in response to external signals. Because the
eukaryotic protein kinases belong to a common superfamily, the structure of the active site and mechanism of phosphorylation are very similar in all of them. Thus protein
kinase A can serve as a general model for this important class
of enzymes.
The active site of protein kinase A is located in the 240residue “ kinase core” of the catalytic subunit. The kinase
core, which is largely conserved in all protein kinases, is responsible for the binding of substrates (ATP and a target peptide sequence) and the subsequent transfer of a phosphate
group from ATP to a serine, threonine, or tyrosine residue
in the target sequence. The kinase core consists of a large domain and small one, with an intervening deep cleft; the active
site comprises residues located in both domains.
Substrate Binding by Protein Kinases The structure of the
ATP-binding site in the catalytic kinase core complements the
structure of the nucleotide substrate. The adenine ring of ATP
sits snugly at the base of the cleft between the large and the
small domains. A highly conserved sequence, Gly-X-Gly-XX-Gly-X-Val (X can be any amino acid), dubbed the “ glycine
lid,” closes over the adenine ring and holds it in position (Figure 3-17a). O ther conserved residues in the binding pocket
stabilize the highly charged phosphate groups.
Although ATP is a common substrate for all protein kinases, the sequence of the target peptide varies among different kinases. The peptide sequence recognized by protein
kinase A is Arg-Arg-X-Ser-Y, where X is any amino acid and
Y is a hydrophobic amino acid. The part of the polypeptide
chain containing the target serine or threonine residue is
bound to a shallow groove in the large domain of the kinase
core. The peptide specificity of protein kinase A is conferred
by several glutamic acid residues in the large domain, which
form salt bridges with the two arginine residues in the target peptide. Different residues determine the specificity of
other protein kinases.
The catalytic core of protein kinase A exists in an “ open”
and “ closed” conformation (Figure 3-17b). In the open conformation, the large and small domains of the core region are
separated enough that substrate molecules can enter and
bind. When the active site is occupied by substrate, the domains move together into the closed position. This change in
tertiary structure, an example of induced fit, brings the target peptide sequence sufficiently close to accept a phosphate
76
CHAPTER 3 • Protein Structure and Function
Glycine lid
(a)
Sm all dom ain
Target
peptide
Nucleotidebinding
pocket
Large dom ain
Glycine lid
(b)
Sm all
dom ain
Active site
Large
dom ain
the active site. In the open position, ATP can enter and bind
the active site cleft; in the closed position, the glycine lid prevents ATP from leaving the cleft. Subsequent to phosphoryl
transfer from the bound ATP to the bound peptide sequence,
the glycine lid must rotate back to the open position before
ADP can be released. Kinetic measurements show that the rate
of ADP release is 20-fold slower than that of phosphoryl transfer, indicating the influence of the glycine lid on the rate of kinase reactions. M utations in the glycine lid that inhibit its
flexibility slow catalysis by protein kinase A even further.
Phosphoryl Transfer by Protein Kinases After substrates have
bound and the catalytic core of protein kinase A has assumed
the closed conformation, the phosphorylation of a serine or
threonine residue on the target peptide can take place (Figure
3-18). As with all chemical reactions, phosphoryl transfer catalyzed by protein kinase A proceeds through a transition state
in which the phosphate group to be transferred and the acceptor hydroxyl group are brought into close proximity. Binding and stabilization of the intermediates by protein kinase A
reduce the activation energy of the phosphoryl transfer reaction, permitting it to take place at measurable rates under the
mild conditions present within cells (see Figure 3-16). Formation of the products induces the enzyme to revert to its open
conformational state, allowing ADP and the phosphorylated
target peptide to diffuse from the active site.
Vmax and Km Characterize an Enzymatic Reaction
Open
Closed
▲ FIGU RE 3 -17 Protein kinase A and conformational
change induced by substrate binding. (a) M odel of the
catalytic subunit of protein kinase A w ith bound substrates; the
conserved kinase core is indicated as a molecular surface. An
overhanging glycine-rich sequence (blue) traps ATP (green) in a
deep cleft between the large and small domains of the core.
Residues in the large domain bind the target peptide (red). The
structure of the kinase core is largely conserved in other
eukaryotic protein kinases. (b) Schematic diagrams of open and
closed conformations of the kinase core. In the absence of
substrate, the kinase core is in the open conformation. Substrate
binding causes a rotation of the large and small domains that
brings the ATP- and peptide-binding sites closer together and
causes the glycine lid to move over the adenine residue of ATP,
thereby trapping the nucleotide in the binding cleft. The model in
part (a) is in the closed conformation.
group from the bound ATP. After the phosphorylation reaction has been completed, the presence of the products causes
the domains to rotate to the open position, from which the
products are released.
The rotation from the open to the closed position also
causes movement of the glycine lid over the ATP-binding cleft.
The glycine lid controls the entry of ATP and release of ADP at
The catalytic action of an enzyme on a given substrate can be
described by two parameters: V max , the maximal velocity of
the reaction at saturating substrate concentrations, and K m
(the Michaelis constant), a measure of the affinity of an enzyme for its substrate (Figure 3-19). The K m is defined as the
substrate concentration that yields a half-maximal reaction
1
rate (i.e., 2 V max ). The smaller the value of K m , the more
avidly an enzyme can bind substrate from a dilute solution
and the smaller the substrate concentration needed to reach
half-maximal velocity.
The concentrations of the various small molecules in a
cell vary widely, as do the K m values for the different enzymes that act on them. Generally, the intracellular concentration of a substrate is approximately the same as or greater
than the K m value of the enzyme to which it binds.
Enzymes in a Common Pathway Are Often
Physically Associated with One Another
Enzymes taking part in a common metabolic process (e.g.,
the degradation of glucose to pyruvate) are generally located
in the same cellular compartment (e.g., in the cytosol, at a
membrane, within a particular organelle). Within a compartment, products from one reaction can move by diffusion
to the next enzyme in the pathway. H owever, diffusion entails random movement and is a slow, inefficient process for
3.3 • Enzymes and the Chemical Work of Cells
(a)
Asp-184
−
Lys-72
+
−
O
O
O
O
Pα
ATP
P
β
−
+ M g 2+
O
O
O
O
M g 2+ +
Rate of form ation of reaction
product (P) (relative units)
Initial state
Asp-166
−
O
P
γ
O H
2−
O
CH2
C
O
P
P
ATP
+ Mg
O
O
P
O
M g 2+ +
2+
O
O
O
O
CH2
C
End state
O
−
O
P
O 2−
P
O
O
O
Vm ax
0.5
[E] = 0.25 unit
0
Km
Vm ax
0.8
High-affinity
substrate
(S)
0.6
Low -affinity
substrate (S’)
0.4
Km for S’
0.2
0
Km for S
Concentration of substrate ([S] or [S’])
▲ EX PERIM EN TA L FIGU RE 3 -19 The Km and V max for an
Phosphate transfer
O
1.0
Concentration of substrate [S]
Rate of reaction
O
[E] = 1.0 unit
1.0
Intermediate state
O
1.5
(b)
Form ation of
transition state
O
Vm ax
2.0
Ser or Thr of
target peptide
+
Lys-168
ADP
77
2−
O
O
P
O
O
CH2
C
Phosphorylated
peptide
▲ FIGU RE 3 -18 M echanism of phosphorylation by protein
kinase A. (Top) Initially, ATP and the target peptide bind to the
active site (see Figure 3-17a). Electrons of the phosphate group
are delocalized by interactions w ith lysine side chains and M g2⫹.
Colored circles represent the residues in the kinase core critical
to substrate binding and phosphoryl transfer. Note that these
residues are not adjacent to one another in the amino acid
sequence. (M iddle) A new bond then forms between the serine
or threonine side-chain oxygen atom and ␥ phosphate, yielding a
pentavalent intermediate. (Bottom) The phosphoester bond
between the  and ␥ phosphates is broken, yielding the products
ADP and a peptide w ith a phosphorylated serine or threonine
side chain. The catalytic mechanism of other protein kinases is
similar.
enzyme-catalyzed reaction are determined from plots of the
initial velocity versus substrate concentration. The shape of
these hypothetical kinetic curves is characteristic of a simple
enzyme-catalyzed reaction in w hich one substrate (S) is
converted into product (P). The initial velocity is measured
immediately after addition of enzyme to substrate before the
substrate concentration changes appreciably. (a) Plots of the
initial velocity at two different concentrations of enzyme [E] as a
function of substrate concentration [S]. The [S] that yields a halfmaximal reaction rate is the M ichaelis constant Km , a measure of
the affinity of E for S. Doubling the enzyme concentration causes
a proportional increase in the reaction rate, and so the maximal
velocity Vmax is doubled; the Km , however, is unaltered. (b) Plots
of the initial velocity versus substrate concentration w ith a
substrate S for w hich the enzyme has a high affinity and w ith a
substrate S⬘ for w hich the enzyme has a low affinity. Note that
the Vmax is the same w ith both substrates but that Km is higher
for S⬘, the low-affinity substrate.
moving molecules between widely dispersed enzymes (Figure
3-20a). To overcome this impediment, cells have evolved
mechanisms for bringing enzymes in a common pathway
into close proximity.
In the simplest such mechanism, polypeptides with different catalytic activities cluster closely together as subunits of
a multimeric enzyme or assemble on a common “ scaffold”
(Figure 3-20b). This arrangement allows the products of one
reaction to be channeled directly to the next enzyme in the
pathway. The first approach is illustrated by pyruvate
78
(a)
CHAPTER 3 • Protein Structure and Function
(a)
Reactants
E1
E2
Products
A
E3
C
B
(b)
(b)
Products
Reactants
A
Reactants
B
Pyruvate
OR
C
O
A
B
HSCoA
CH3C
COO−
E1
O
CO2
Scaffold
Products
E2
C
CH3C
SCoA
E3
Acetyl CoA
(c)
Reactants
NAD+
Products
A
B
Net reaction:
Pyruvate + NAD+ + CoA
C
▲ FIGU RE 3 -2 0 Evolution of multifunctional enzyme.
In the hypothetical reaction pathways illustrated here the initial
reactants are converted into final products by the sequential
action of three enzymes: A, B, and C. (a) When the enzymes are
free in solution or even constrained w ithin the same cellular
compartment, the intermediates in the reaction sequence must
diffuse from one enzyme to the next, an inherently slow process.
(b) Diffusion is greatly reduced or eliminated w hen the enzymes
associate into multisubunit complexes. (c) The closest integration
of different catalytic activities occurs w hen the enzymes are
fused at the genetic level, becoming domains in a single protein.
dehydrogenase, a complex of three distinct enzymes that converts pyruvate into acetyl CoA in mitochondria (Figure 3-21).
The scaffold approach is employed by M AP kinase signaltransduction pathways, discussed in Chapter 14. In yeast,
three protein kinases assembled on the Ste5 scaffold protein
form a kinase cascade that transduces the signal triggered by
the binding of mating factor to the cell surface.
In some cases, separate proteins have been fused together
at the genetic level to create a single multidomain, multifunctional enzyme (Figure 3-20c). For instance, the isomerization of citrate to isocitrate in the citric acid cycle is
catalyzed by aconitase, a single polypeptide that carries out
two separate reactions: (1) the dehydration of citrate to form
cis-aconitate and then (2) the hydration of cis-aconitate to
yield isocitrate (see Figure 8-9).
K EY C O N C EP T S O F S EC T I O N 3 . 3
Enzymes and the Chemical Work of Cells
■ The function of nearly all proteins depends on their ability to bind other molecules (ligands). Ligand-binding sites
NADH +
H+
CO2 + NADH + acetyl CoA
▲ FIGU RE 3 -2 1 Structure and function of pyruvate
dehydrogenase, a large multimeric enzyme complex that
converts pyruvate into acetyl CoA. (a) The complex consists of
24 copies of pyruvate decarboxylase (E1), 24 copies of lipoamide
transacetylase (E2), and 12 copies of dihydrolipoyl dehydrogenase
(E3). The E1 and E3 subunits are bound to the outside of the core
formed by the E2 subunits. (b) The reactions catalyzed by the
complex include several enzyme-bound intermediates (not
show n). The tight structural integration of the three enzymes
increases the rate of the overall reaction and minimizes possible
side reactions.
on proteins and the corresponding ligands are chemically
and topologically complementary.
The affinity of a protein for a particular ligand refers to
the strength of binding; its specificity refers to the preferential binding of one or a few closely related ligands.
■
Enzymes are catalytic proteins that accelerate the rate
of cellular reactions by lowering the activation energy
and stabilizing transition-state intermediates (see Figure
3-16).
■
An enzyme active site comprises two functional parts: a
substrate-binding region and a catalytic region. The amino
acids composing the active site are not necessarily adjacent
in the amino acid sequence but are brought into proximity in the native conformation.
■
■ From plots of reaction rate versus substrate concentration, two characteristic parameters of an enzyme can
be determined: the M ichaelis constant K m , a measure of
the enzyme’s affinity for substrate, and the maximal velocity V max , a measure of its catalytic power (see Figure
3-19).
3.4 • Molecular Motors and the Mechanical Work of Cells
■ Enzymes in a common pathway are located within specific cell compartments and may be further associated as
domains of a monomeric protein, subunits of a multimeric
protein, or components of a protein complex assembled on
a common scaffold (see Figure 3-20).
3.4 Molecular Motors and
the Mechanical Work of Cells
A common property of all cells is motility, the ability to move
in a specified direction. Many cell processes exhibit some type of
movement at either the molecular or the cellular level; all movements result from the application of a force. In Brownian motion, for instance, thermal energy constantly buffets molecules
and organelles in random directions and for very short distances. O n the other hand, materials within a cell are transported in specific directions and for longer distances. This type
of movement results from the mechanical work carried out by
proteins that function as motors. We first briefly describe the
types and general properties of molecular motors and then look
at how one type of motor protein generates force for movement.
Molecular Motors Convert Energy into Motion
At the nanoscale of cells and molecules, movement is effected
by much different forces from those in the macroscopic world.
For example, the high protein concentration (200–300 mg/ml)
of the cytoplasm prevents organelles and vesicles from diffusing faster than 100 m/3 hours. Even a micrometer-sized bacterium experiences a drag force from water that stops its
forward movement within a fraction of a nanometer when it
stops actively swimming. To generate the forces necessary for
many cellular movements, cells depend on specialized enzymes
commonly called motor proteins. These mechanochemical enzym es convert energy released by the hydrolysis of ATP or
from ion gradients into a mechanical force.
M otor proteins generate either linear or rotary motion
(Table 3-2). Some motor proteins are components of macro(a)
molecular assemblies, but those that move along cytoskeletal
fibers are not. This latter group comprises the myosins, kinesins, and dyneins—linear motor proteins that carry attached “ cargo” with them as they proceed along either
microfilaments or microtubules (Figure 3-22a). DN A and
RN A polymerases also are linear motor proteins because
they translocate along DN A during replication and transcription. In contrast, rotary motors revolve to cause the beat
of bacterial flagella, to pack DN A into the capsid of a virus,
and to synthesize ATP. The propulsive force for bacterial
swimming, for instance, is generated by a rotary motor protein complex in the bacterial membrane. Ions flow down an
electrochemical gradient through an immobile ring of proteins, the stator, which is located in the membrane. Torque
generated by the stator rotates an inner ring of proteins and
the attached flagellum (Figure 3-22b). Similarly, in the mitochondrial ATP synthase, or F0 F1 complex, a flux of ions
across the inner mitochondrial membrane is transduced by
the F0 part into rotation of the ␥ subunit, which projects into
a surrounding ring of ␣ and  subunits in the F1 part. Interactions between the ␥ subunit and the  subunits directs the
synthesis of ATP (Chapter 8).
From the observed activities of motor proteins, we can
infer three general properties that they possess:
The ability to transduce a source of energy, either ATP
or an ion gradient, into linear or rotary movement
■
The ability to bind and translocate along a cytoskeletal
filament, nucleic acid strand, or protein complex
■
■
N et movement in a given direction
The motor proteins that attach to cytoskeletal fibers also
bind to and carry along cargo as they translocate. The cargo
in muscle cells and eukaryotic flagella consists of thick filaments and B tubules, respectively (see Figure 3-22a). These
motor proteins can also transport cargo chromosomes and
membrane-limited vesicles as they move along microtubules
or microfilaments (Figure 3-23).
(b)
Flagellum
ADP
M yosin
or
dynein
Ions
Actin filam ent or A tubule
Rotor
▲ FIGU RE 3 -2 2 Comparison of linear and rotary molecular
motors. (a) In muscle and eukaryotic flagella, the head domains
of motor proteins (blue) bind to an actin thin filament (muscle) or
the A tubule of a doublet microtubule (flagella). ATP hydrolysis in
the head causes linear movement of the cytoskeletal fiber
(orange) relative to the attached thick filament or B tubule of an
adjacent doublet microtubule. (b) In the rotary motor in
the bacterial membrane, the stator (blue) is immobile in
the membrane. Ion flow through the stator generates a
torque that powers rotation of the rotor (orange) and the
flagellum attached to it.
M EDIA CON N ECTION S
Stator
Video: Rotary Motor Action:
Flagellum
Thick filam ent or B tubule
ATP
79
80
CHAPTER 3 • Protein Structure and Function
TABLE 3-2
Selected Molecular Motors
Motor*
Energy
Source
Structure/ Components
Cellular Location
Movement Generated
LIN EAR M O TO RS
DN A polymerase (4)
ATP
M ultisubunit polymerase ␦
within replisome
N ucleus
Translocation along DN A
during replication
RN A polymerase (4)
ATP
M ultisubunit polymerase
within transcription
elongation complex
N ucleus
Translocation along DN A
during transcription
Ribosome (4)
GTP
Elongation factor 2 (EF2)
bound to ribosome
Cytoplasm/ER
membrane
Translocation along mRN A
during translation
M yosins (3, 19)
ATP
H eavy and light chains;
head domains with ATPase
activity and microfilamentbinding site
Cytoplasm
Transport of cargo
vesicles; contraction
Kinesins (20)
ATP
H eavy and light chains; head
domains with ATPase activity
and microtubule-binding site
Cytoplasm
Transport of cargo
vesicles and chromosomes
during mitosis
Dyneins (20)
ATP
M ultiple heavy, intermediate,
and light chains; head domains
with ATPase activity and
microtubule-binding site
Cytoplasm
Transport of cargo
vesicles; beating of cilia
and eukaryotic flagella
Bacterial flagellar
motor
H ⫹/N a ⫹
gradient
Stator and rotor proteins,
flagellum
Plasma membrane
Rotation of flagellum
attached to rotor
ATP synthase,
F0 F1 (8)
H⫹
gradient
M ultiple subunits forming
F0 and F1 particles
Inner mitochondrial
membrane, thylakoid
membrane, bacterial
plasma membrane
Rotation of ␥ subunit
leading to ATP synthesis
Viral capsid motor
ATP
Connector, prohead
RN A, ATPase
Capsid
Rotation of connector
leading to DN A packaging
R O TARY M O TO RS
*
N umbers in parentheses indicate chapters in which various motors are discussed.
Cargo
Cargo binding
䉴 FIGU RE 3 -2 3 M otor protein-dependent movement of
cargo. The head domains of myosin, dynein, and kinesin motor
proteins bind to a cytoskeletal fiber (microfilaments or
microtubules), and the tail domain attaches to one of various
types of cargo—in this case, a membrane-limited vesicle.
Hydrolysis of ATP in the head domain causes the head domain to
“ walk” along the track in one direction by a repeating cycle of
conformational changes.
Tail
Neck
M otor
protein
ATP hydrolysis
Fiber binding
Head
Cytoskeletal fiber
3.4 • Molecular Motors and the Mechanical Work of Cells
81
(b) Head dom ain
(a) M yosin II
Tail
Head Neck
Nucleotidebinding site
Regulatory
light chain
Essential
light chain
Heavy chains
Regulatory
light chain
Actinbinding
site
Essential
light chain
Heavy chain
▲ FIGU RE 3 -2 4 Structure of myosin II. (a) M yosin II is a
dimeric protein composed of two identical heavy chains (w hite)
and four light chains (blue and green). Each of the head domains
transduces the energy from ATP hydrolysis into movement. Two
light chains are associated w ith the neck domain of each heavy
chain. The coiled-coil sequence of the tail domain organizes
myosin II into a dimer. (b) Three-dimensional model of a single
head domain show s that it has a curved, elongated shape and is
bisected by a large cleft. The nucleotide-binding pocket lies on
one side of this cleft, and the actin-binding site lies on the other
side near the tip of the head. Wrapped around the shaft of the ␣helical neck are the two light chains. These chains stiffen the
neck so that it can act as a lever arm for the head. Show n here
is the ADP-bound conformation.
All Myosins Have Head, Neck, and Tail Domains
with Distinct Functions
head, wrapped around the neck like C-clamps. In this position, the light chains stiffen the neck region and are therefore
able to regulate the activity of the head domain.
To further illustrate the properties of motor proteins, we consider myosin II, which moves along actin filaments in muscle
cells during contraction. O ther types of myosin can transport
vesicles along actin filaments in the cytoskeleton. M yosin II
and other members of the myosin superfamily are composed
of one or two heavy chains and several light chains. The
heavy chains are organized into three structurally and functionally different types of domains (Figure 3-24a).
The two globular head dom ains are specialized ATPases
that couple the hydrolysis of ATP with motion. A critical feature of the myosin ATPase activity is that it is actin activated.
In the absence of actin, solutions of myosin slowly convert
ATP into ADP and phosphate. H owever, when myosin is
complexed with actin, the rate of myosin ATPase activity is
four to five times as fast as it is in the absence of actin. The
actin-activation step ensures that the myosin ATPase operates at its maximal rate only when the myosin head domain is bound to actin. Adjacent to the head domain lies the
␣-helical neck region, which is associated with the light
chains. These light chains are crucial for converting small
conformational changes in the head into large movements
of the molecule and for regulating the activity of the head domain. The rodlike tail dom ain contains the binding sites that
determine the specific activities of a particular myosin.
The results of studies of myosin fragments produced by
proteolysis helped elucidate the functions of the domains.
X-ray crystallographic analysis of the S1 fragment of myosin
II, which consists of the head and neck domains, revealed its
shape, the positions of the light chains, and the locations of
the ATP-binding and actin-binding sites. The elongated
myosin head is attached at one end to the ␣-helical neck (Figure 3-24b). Two light-chain molecules lie at the base of the
Conformational Changes in the Myosin Head
Couple ATP Hydrolysis to Movement
The results of studies of muscle contraction provided the first
evidence that myosin heads slide or walk along actin filaments. Unraveling the mechanism of muscle contraction
was greatly aided by the development of in vitro motility assays and single-molecule force measurements. O n the basis
of information obtained with these techniques and the threedimensional structure of the myosin head, researchers developed a general model for how myosin harnesses the energy
released by ATP hydrolysis to move along an actin filament.
Because all myosins are thought to use the same mechanism
to generate movement, we will ignore whether the myosin
tail is bound to a vesicle or is part of a thick filament as it is
in muscle. O ne assumption in this model is that the hydrolysis of a single ATP molecule is coupled to each step taken by
a myosin molecule along an actin filament. Evidence supporting this assumption is discussed in Chapter 19.
As shown in Figure 3-25, myosin undergoes a series of
events during each step of movement. In the course of one
cycle, myosin must exist in at least three conformational
states: an ATP state unbound to actin, an ADP-P i state
bound to actin, and a state after the power-generating
stroke has been completed. The major question is how the
nucleotide-binding pocket and the distant actin-binding site
are mutually influenced and how changes at these sites are
converted into force. The results of structural studies of
myosin in the presence of nucleotides and nucleotide
analogs that mimic the various steps in the cycle indicate
that the binding and hydrolysis of a nucleotide cause a
82
CHAPTER 3 • Protein Structure and Function
Thick filam ent
ATP-binding
site
M yosin head
Actin thin filam ent
Nucleotide
binding
1
ATP
Head dissociates
from filam ent
Hydrolysis
2
䉳 FIGU RE 3 -2 5 Operational model for the coupling of ATP
hydrolysis to movement of myosin along an actin filament.
Show n here is the cycle for a myosin II head that is part of a
thick filament in muscle, but other myosins that attach to other
cargo (e.g., the membrane of a vesicle) are thought to operate
according to the same cyclical mechanism. In the absence of
bound nucleotide, a myosin head binds actin tightly in a “ rigor”
state. Step 1 : Binding of ATP opens the cleft in the myosin
head, disrupting the actin-binding site and weakening the
interaction w ith actin. Step 2 : Freed of actin, the myosin head
hydrolyzes ATP, causing a conformational change in the head that
moves it to a new position, closer to the (⫹) end of the actin
filament, w here it rebinds to the filament. Step 3 : As phosphate
(Pi) dissociates from the ATP-binding pocket, the myosin head
undergoes a second conformational change—the power stroke—
w hich restores myosin to its rigor conformation. Because myosin
is bound to actin, this conformational change exerts a force that
causes myosin to move the actin filament. Step 4 : Release of
ADP completes the cycle. [Adapted from R. D. Vale and R. A. M illigan,
2002, Science 288:88.]
Head pivots and
binds a new
actin subunit
Focus Animation: Myosin Crossbridge Cycle
M EDIA CON N ECTION S
ADP•Pi
K EY C O N C EP T S O F S EC T I O N 3 . 4
Molecular Motors and the Mechanical Work of Cells
Pi release
M otor proteins are mechanochemical enzymes that convert energy released by ATP hydrolysis into either linear
or rotary movement (see Figure 3-22).
■
3
Pi
ADP
Head pivots and
m oves filam ent
(pow er stroke)
Linear motor proteins (myosins, kinesins, and dyneins)
move along cytoskeletal fibers carrying bound cargo,
which includes vesicles, chromosomes, thick filaments in
muscle, and microtubules in eukaryotic flagella.
■
M yosin II consists of two heavy chains and several light
chains. Each heavy chain has a head (motor) domain,
which is an actin-activated ATPase; a neck domain, which
is associated with light chains; and a long rodlike tail domain that organizes the dimeric molecule and binds to thick
filaments in muscle cells (see Figure 3-24).
■
ADP release
4
ADP
small conformational change in the head domain that is
amplified into a large movement of the neck region. The
small conformational change in the head domain is localized to a “ switch” region consisting of the nucleotide- and
actin-binding sites. A “ converter” region at the base of the
head acts like a fulcrum that causes the leverlike neck to
bend and rotate.
H omologous switch, converter, and lever arm structures
in kinesin are responsible for the movement of kinesin motor
proteins along microtubules. The structural basis for dynein
movement is unknown because the three-dimensional structure of dynein has not been determined.
M ovement of myosin relative to an actin filament results
from the attachment of the myosin head to an actin filament, rotation of the neck region, and detachment in a
cyclical ATP-dependent process (see Figure 3-25). The same
general mechanism is thought to account for all myosinand kinesin-mediated movement.
■
3.5 Common Mechanisms
for Regulating Protein Function
M ost processes in cells do not take place independently of
one another or at a constant rate. Instead, the catalytic activity of enzymes or the assembly of a macromolecular complex is so regulated that the amount of reaction product or
the appearance of the complex is just sufficient to meet the
needs of the cell. As a result, the steady-state concentrations
3.5 • Common Mechanisms for Regulating Protein Function
of substrates and products will vary, depending on cellular
conditions. The flow of material in an enzymatic pathway is
controlled by several mechanisms, some of which also regulate the functions of nonenzymatic proteins.
O ne of the most important mechanisms for regulating
protein function entails allostery. Broadly speaking, allostery
refers to any change in a protein’s tertiary or quaternary
structure or both induced by the binding of a ligand, which
may be an activator, inhibitor, substrate, or all three. Allosteric regulation is particularly prevalent in multimeric enzymes and other proteins. We first explore several ways in
which allostery influences protein function and then consider
other mechanisms for regulating proteins.
Cooperative Binding Increases a Protein’s
Response to Small Changes in Ligand
Concentration
In many cases, especially when a protein binds several molecules of one ligand, the binding is graded; that is, the binding of one ligand molecule affects the binding of subsequent
ligand molecules. This type of allostery, often called cooper-
% Saturation
100
50
P50 = 26
0
20
40
60
p O2 (torr)
p O2 in capillaries
of active m uscles
80
100
p O2 in alveoli
of lungs
▲ EX PERIM EN TA L FIGU RE 3 -2 6 Sequential binding of
oxygen to hemoglobin exhibits positive cooperativity. Each
hemoglobin molecule has four oxygen-binding sites; at saturation
all the sites are loaded w ith oxygen. The oxygen concentration is
commonly measured as the partial pressure (pO2). P50 is the pO2
at w hich half the oxygen-binding sites at a given hemoglobin
concentration are occupied; it is equivalent to the Km for an
enzymatic reaction. The large change in the amount of oxygen
bound over a small range of pO2 values permits efficient
unloading of oxygen in peripheral tissues such as muscle. The
sigmoidal shape of a plot of percent saturation versus ligand
concentration is indicative of cooperative binding. In the absence
of cooperative binding, a binding curve is a hyperbola, similar to
the simple kinetic curves in Figure 3-19. [Adapted from L. Stryer,
Biochemistry, 4th ed., 1995, W. H. Freeman and Company.]
83
ativity, permits many multisubunit proteins to respond more
efficiently to small changes in ligand concentration than
would otherwise be possible. In positive cooperativity, sequential binding is enhanced; in negative cooperativity,
sequential binding is inhibited.
H emoglobin presents a classic example of positive cooperative binding. Each of the four subunits in hemoglobin
contains one heme molecule, which consists of an iron atom
held within a porphyrin ring (see Figure 8-16a). The heme
groups are the oxygen-binding components of hemoglobin
(see Figure 3-10). The binding of oxygen to the heme molecule in one of the four hemoglobin subunits induces a local
conformational change whose effect spreads to the other
subunits, lowering the K m for the binding of additional oxygen molecules and yielding a sigmoidal oxygen-binding curve
(Figure 3-26). Consequently, the sequential binding of oxygen is facilitated, permitting hemoglobin to load more oxygen in peripheral tissues than it otherwise could at normal
oxygen concentrations.
Ligand Binding Can Induce Allosteric Release
of Catalytic Subunits or Transition to a State
with Different Activity
Previously, we looked at protein kinase A to illustrate binding and catalysis by the active site of an enzyme. This enzyme
can exist as an inactive tetrameric protein composed of two
catalytic subunits and two regulatory subunits. Each regulatory subunit contains a pseudosubstrate sequence that binds
to the active site in a catalytic subunit. By blocking substrate
binding, the regulatory subunit inhibits the activity of the
catalytic subunit.
Inactive protein kinase A is turned on by cyclic AMP
(cAMP), a small second-messenger molecule. The binding of
cAM P to the regulatory subunits induces a conformational
change in the pseudosubstrate sequence so that it can no
longer bind the catalytic subunit. Thus, in the presence of
cAM P, the inactive tetramer dissociates into two monomeric
active catalytic subunits and a dimeric regulatory subunit
(Figure 3-27). As discussed in Chapter 13, the binding of various hormones to cell-surface receptors induces a rise in the
intracellular concentration of cAM P, leading to the activation of protein kinase A. When the signaling ceases and the
cAM P level decreases, the activity of protein kinase A is
turned off by reassembly of the inactive tetramer. The binding of cAM P to the regulatory subunits exhibits positive cooperativity; thus small changes in the concentration of this
allosteric molecule produce a large change in the activity of
protein kinase A.
M any multimeric enzymes undergo allosteric transitions
that alter the relation of the subunits to one another but do
not cause dissociation as in protein kinase A. In this type
of allostery, the activity of a protein in the ligand-bound
state differs from that in the unbound state. An example is
the GroEL chaperonin discussed earlier. This barrel-shaped
84
CHAPTER 3 • Protein Structure and Function
(a)
Catalytic site
Nucleotidebinding site
Pseudosubstrate
C
C
+
C
R
R
C
+
Inactive PKA
R
R
Active PKA
cAM P
NH2
(b)
C
N
C
HC
C
N
CH
N
O
CH2
H
O
H
H
P
O
ⴚ
O
N
H
O
OH
cyclic AM P
(cAM P)
▲ FIGU RE 3 -2 7 Ligand-induced activation of protein kinase
A (PKA). At low concentrations of cyclic AM P (cAM P), the PKA
is an inactive tetramer. Binding of cAM P to the regulatory (R)
subunits causes a conformational change in these subunits that
permits release of the active, monomeric catalytic (C) subunits.
(b) Cyclic AM P is a derivative of adenosine monophosphate. This
intracellular signaling molecule, w hose concentration rises in
response to various extracellular signals, can modulate the
activity of many proteins.
100-fold by the release of Ca 2⫹ from ER stores or by its import from the extracellular environment. This rise in cytosolic Ca 2⫹ is sensed by Ca 2⫹-binding proteins, particularly
those of the EF hand fam ily, all of which contain the helixloop-helix motif discussed earlier (see Figure 3-6a).
The prototype EF hand protein, calmodulin, is found in
all eukaryotic cells and may exist as an individual
monomeric protein or as a subunit of a multimeric protein. A
dumbbell-shaped molecule, calmodulin contains four Ca 2⫹binding sites with a K D of ≈10 ⫺6 M . The binding of Ca 2⫹ to
calmodulin causes a conformational change that permits
Ca 2⫹/calmodulin to bind various target proteins, thereby
switching their activity on or off (Figure 3-28). Calmodulin
and similar EF hand proteins thus function as sw itch proteins, acting in concert with Ca 2⫹ to modulate the activity
of other proteins.
Switching Mediated by Guanine Nucleotide–Binding
Proteins Another group of intracellular switch proteins constitutes the GTPase superfamily. These proteins include
monomeric Ras protein (see Figure 3-5) and the G ␣ subunit of
the trimeric G proteins. Both Ras and G ␣ are bound to the
plasma membrane, function in cell signaling, and play a key
role in cell proliferation and differentiation. O ther members
EF1
EF3
EF2
protein-folding machine comprises two back-to-back multisubunit rings, which can exist in a “ tight” peptide-binding
state and a “ relaxed” peptide-releasing state (see Figure
3-11). The binding of ATP and the co-chaperonin GroES to
one of the rings in the tight state causes a twofold expansion
of the GroEL cavity, shifting the equilibrium toward the relaxed peptide-folding state.
EF4
Target
peptide
Ca2+
Calcium and GTP Are Widely Used to Modulate
Protein Activity
In the preceding examples, oxygen, cAM P, and ATP cause allosteric changes in the activity of their target proteins (hemoglobin, protein kinase A, and GroEL, respectively). Two
additional allosteric ligands, Ca 2⫹ and GTP, act through two
types of ubiquitous proteins to regulate many cellular
processes.
Calmodulin-Mediated Switching The concentration of
Ca 2⫹ free in the cytosol is kept very low (≈10 ⫺7 M ) by membrane transport proteins that continually pump Ca 2⫹ out of
the cell or into the endoplasmic reticulum. As we learn in
Chapter 7, the cytosolic Ca 2⫹ level can increase from 10- to
▲ FIGU RE 3 -2 8 Sw itching mediated by Ca2⫹/ calmodulin.
Calmodulin is a w idely distributed cytosolic protein that contains
four Ca2⫹-binding sites, one in each of its EF hands. Each EF
hand has a helix-loop-helix motif. At cytosolic Ca2+ concentrations
above about 5 ⫻ 10⫺7 M , binding of Ca2⫹ to calmodulin changes
the protein’s conformation. The resulting Ca2⫹/calmodulin w raps
around exposed helices of various target proteins, thereby
altering their activity.
3.5 • Common Mechanisms for Regulating Protein Function
Active
Active ("on")
R
GTPase
GDP
GEFs
G
T
P
+
+
+
−
GAPs
RGSs
GDIs
GTPase
G
D
P
OH
Pi
ATP
Protein
phosphatase
Protein
kinase
Inactive ("off ")
GTP
85
O
H2O
R
O
P
ADP
O−
O−
Inactive
▲ FIGU RE 3 -2 9 Cycling of GTPase sw itch proteins between
the active and inactive forms. Conversion of the active into the
inactive form by hydrolysis of the bound GTP is accelerated by
GAPs (GTPase-accelerating proteins) and RGSs (regulators of G
protein–signaling) and inhibited by GDIs (guanine nucleotide
dissociation inhibitors). Reactivation is promoted by GEFs
(guanine nucleotide–exchange factors).
▲ FIGU RE 3 -3 0 Regulation of protein activity by
kinase/ phosphatase sw itch. The cyclic phosphorylation and
dephosphorylation of a protein is a common cellular mechanism
for regulating protein activity. In this example, the target protein
R is inactive (light orange) w hen phosphorylated and active (dark
orange) w hen dephosphorylated; some proteins have the
opposite pattern.
of the GTPase superfamily function in protein synthesis, the
transport of proteins between the nucleus and the cytoplasm,
the formation of coated vesicles and their fusion with target
membranes, and rearrangements of the actin cytoskeleton.
All the GTPase switch proteins exist in two forms (Figure
3-29): (1) an active (“ on” ) form with bound GTP (guanosine
triphosphate) that modulates the activity of specific target
proteins and (2) an inactive (“ off” ) form with bound GDP
(guanosine diphosphate). The GTPase activity of these
switch proteins hydrolyzes bound GTP to GDP slowly, yielding the inactive form. The subsequent exchange of GDP with
GTP to regenerate the active form occurs even more slowly.
Activation is temporary and is enhanced or depressed by
other proteins acting as allosteric regulators of the switch
protein. We examine the role of various GTPase switch proteins in regulating intracellular signaling and other processes
in several later chapters.
Nearly 3 percent of all yeast proteins are protein kinases or
phosphatases, indicating the importance of phosphorylation
and dephosphorylation reactions even in simple cells. All
classes of proteins—including structural proteins, enzymes,
membrane channels, and signaling molecules—are regulated
by kinase/phosphatase switches. Different protein kinases and
phosphatases are specific for different target proteins and can
thus regulate a variety of cellular pathways, as discussed in
later chapters. Some of these enzymes act on one or a few target proteins, whereas others have multiple targets. The latter
are useful in integrating the activities of proteins that are coordinately controlled by a single kinase/phosphatase switch.
Frequently, another kinase or phosphatase is a target, thus creating a web of interdependent controls.
Cyclic Protein Phosphorylation
and Dephosphorylation Regulate
Many Cellular Functions
The regulatory mechanisms discussed so far act as switches,
reversibly turning proteins on and off. The regulation of
some proteins is by a distinctly different mechanism: the irreversible activation or inactivation of protein function by
proteolytic cleavage. This mechanism is most common in regard to some hormones (e.g., insulin) and digestive proteases. Good examples of such enzymes are trypsin and
chymotrypsin, which are synthesized in the pancreas and secreted into the small intestine as the inactive zymogens
trypsinogen and chym otrypsinogen, respectively. Enterokinase, an aminopeptidase secreted from cells lining the small
intestine, converts trypsinogen into trypsin, which in turn
cleaves chymotrypsinogen to form chymotrypsin. The delay
in the activation of these proteases until they reach the intestine prevents them from digesting the pancreatic tissue in
which they are made.
As noted earlier, one of the most common mechanisms for
regulating protein activity is phosphorylation, the addition
and removal of phosphate groups from serine, threonine, or
tyrosine residues. Protein kinases catalyze phosphorylation,
and phosphatases catalyze dephosphorylation. Although
both reactions are essentially irreversible, the counteracting
activities of kinases and phosphatases provide cells with a
“ switch” that can turn on or turn off the function of various proteins (Figure 3-30). Phosphorylation changes a protein’s charge and generally leads to a conformational change;
these effects can significantly alter ligand binding by a protein, leading to an increase or decrease in its activity.
Proteolytic Cleavage Irreversibly Activates
or Inactivates Some Proteins
86
CHAPTER 3 • Protein Structure and Function
Higher-Order Regulation Includes Control
of Protein Location and Concentration
activity state into another or to the release of active subunits (see Figure 3-27).
The activities of proteins are extensively regulated in order
that the numerous proteins in a cell can work together harmoniously. For example, all metabolic pathways are closely
controlled at all times. Synthetic reactions take place when
the products of these reactions are needed; degradative reactions take place when molecules must be broken down.
All the regulatory mechanisms heretofore described affect a
protein locally at its site of action, turning its activity on
or off.
N ormal functioning of a cell, however, also requires the
segregation of proteins to particular compartments such as
the mitochondria, nucleus, and lysosomes. In regard to enzymes, compartmentation not only provides an opportunity
for controlling the delivery of substrate or the exit of product
but also permits competing reactions to take place simultaneously in different parts of a cell. We describe the mechanisms that cells use to direct various proteins to different
compartments in Chapters 16 and 17.
In addition to compartmentation, cellular processes are
regulated by protein synthesis and degradation. For example,
proteins are often synthesized at low rates when a cell has little or no need for their activities. When the cell faces increased demand (e.g., appearance of substrate in the case of
enzymes, stimulation of B lymphocytes by antigen), the cell
responds by synthesizing new protein molecules. Later, the
protein pool is lowered when levels of substrate decrease or
the cell becomes inactive. Extracellular signals are often instrumental in inducing changes in the rates of protein synthesis and degradation (Chapters 13–15). Such regulated
changes play a key role in the cell cycle (Chapter 21) and in
cell differentiation (Chapter 22).
Two classes of intracellular switch proteins regulate a
variety of cellular processes: (1) calmodulin and related
Ca 2⫹-binding proteins in the EF hand family and (2) members of the GTPase superfamily (e.g., Ras and G ␣), which
cycle between active GTP-bound and inactive GDP-bound
forms (see Figure 3-29).
K EY C O N C EP T S O F S EC T I O N 3 . 5
Common Mechanisms for Regulating
Protein Function
■ In allostery, the binding of one ligand molecule (a substrate, activator, or inhibitor) induces a conformational
change, or allosteric transition, that alters a protein’s activity or affinity for other ligands.
■ In multimeric proteins, such as hemoglobin, that bind
multiple ligand molecules, the binding of one ligand molecule may modulate the binding affinity for subsequent ligand molecules. Enzymes that cooperatively bind substrates
exhibit sigmoidal kinetics similar to the oxygen-binding
curve of hemoglobin (see Figure 3-26).
■
The phosphorylation and dephosphorylation of amino
acid side chains by protein kinases and phosphatases provide reversible on/off regulation of numerous proteins.
■
N onallosteric mechanisms for regulating protein activity include proteolytic cleavage, which irreversibly converts
inactive zymogens into active enzymes, compartmentation
of proteins, and signal-induced modulation of protein synthesis and degradation.
■
3.6 Purifying, Detecting,
and Characterizing Proteins
A protein must be purified before its structure and the
mechanism of its action can be studied. H owever, because
proteins vary in size, charge, and water solubility, no single
method can be used to isolate all proteins. To isolate one
particular protein from the estimated 10,000 different proteins in a cell is a daunting task that requires methods both
for separating proteins and for detecting the presence of specific proteins.
Any molecule, whether protein, carbohydrate, or nucleic
acid, can be separated, or resolved, from other molecules on
the basis of their differences in one or more physical or
chemical characteristics. The larger and more numerous the
differences between two proteins, the easier and more efficient their separation. The two most widely used characteristics for separating proteins are size, defined as either length
or mass, and binding affinity for specific ligands. In this section, we briefly outline several important techniques for separating proteins; these techniques are also useful for the
separation of nucleic acids and other biomolecules. (Specialized methods for removing membrane proteins from membranes are described in the next chapter after the unique
properties of these proteins are discussed.) We then consider
general methods for detecting, or assaying, specific proteins,
including the use of radioactive compounds for tracking
biological activity. Finally, we consider several techniques
for characterizing a protein’s mass, sequence, and threedimensional structure.
■ Several allosteric mechanisms act as switches, turning
protein activity on and off in a reversible fashion.
Centrifugation Can Separate Particles and
Molecules That Differ in Mass or Density
■ The binding of allosteric ligand molecules may lead to
the conversion of a protein from one conformational/
The first step in a typical protein purification scheme is
centrifugation. The principle behind centrifugation is that
3.6 • Purifying, Detecting, and Characterizing Proteins
two particles in suspension (cells, organelles, or molecules) with different masses or densities will settle to the
bottom of a tube at different rates. Remember, m ass is the
weight of a sample (measured in grams), whereas density
is the ratio of its weight to volume (grams/liter). Proteins
vary greatly in mass but not in density. Unless a protein
has an attached lipid or carbohydrate, its density will not
vary by more than 15 percent from 1.37 g/cm 3 , the average protein density. H eavier or more dense molecules settle, or sediment, more quickly than lighter or less dense
molecules.
A centrifuge speeds sedimentation by subjecting particles
in suspension to centrifugal forces as great as 1,000,000
times the force of gravity g, which can sediment particles as
small as 10 kDa. M odern ultracentrifuges achieve these
forces by reaching speeds of 150,000 revolutions per minute
(rpm) or greater. H owever, small particles with masses of
5 kDa or less will not sediment uniformly even at such high
rotor speeds.
Centrifugation is used for two basic purposes: (1) as a
preparative technique to separate one type of material from
others and (2) as an analytical technique to measure physical properties (e.g., molecular weight, density, shape, and
equilibrium binding constants) of macromolecules. The sedim entation constant, s, of a protein is a measure of its sedimentation rate. The sedimentation constant is commonly
expressed in svedbergs (S): 1 S ⫽ 10 ⫺13 seconds.
Differential Centrifugation The most common initial step in
protein purification is the separation of soluble proteins from
insoluble cellular material by differential centrifugation. A
starting mixture, commonly a cell homogenate, is poured
into a tube and spun at a rotor speed and for a period of time
that forces cell organelles such as nuclei to collect as a pellet
at the bottom; the soluble proteins remain in the supernatant
(Figure 3-31a). The supernatant fraction then is poured off
and can be subjected to other purification methods to separate the many different proteins that it contains.
Rate-Zonal Centrifugation O n the basis of differences in
their masses, proteins can be separated by centrifugation
through a solution of increasing density called a density gradient. A concentrated sucrose solution is commonly used to
form density gradients. When a protein mixture is layered on
top of a sucrose gradient in a tube and subjected to centrifugation, each protein in the mixture migrates down the tube
at a rate controlled by the factors that affect the sedimentation constant. All the proteins start from a thin zone at the
top of the tube and separate into bands, or zones (actually
disks), of proteins of different masses. In this separation technique, called rate-zonal centrifugation, samples are centrifuged just long enough to separate the molecules of
interest into discrete zones (Figure 3-31b). If a sample is centrifuged for too short a time, the different protein molecules
will not separate sufficiently. If a sample is centrifuged much
87
longer than necessary, all the proteins will end up in a pellet
at the bottom of the tube.
Although the sedimentation rate is strongly influenced by
particle mass, rate-zonal centrifugation is seldom effective
in determining precise molecular weights because variations
in shape also affect sedimentation rate. The exact effects of
shape are hard to assess, especially for proteins and singlestranded nucleic acid molecules that can assume many complex shapes. N evertheless, rate-zonal centrifugation has
proved to be the most practical method for separating many
different types of polymers and particles. A second densitygradient technique, called equilibrium density-gradient centrifugation, is used mainly to separate DN A or organelles
(see Figure 5-37).
Electrophoresis Separates Molecules on the Basis
of Their Charge :Mass Ratio
Electrophoresis is a technique for separating molecules in a
mixture under the influence of an applied electric field. Dissolved molecules in an electric field move, or migrate, at a
speed determined by their charge:mass ratio. For example,
if two molecules have the same mass and shape, the one with
the greater net charge will move faster toward an electrode.
SDS-Polyacrylamide Gel Electrophoresis Because many
proteins or nucleic acids that differ in size and shape have
nearly identical charge:mass ratios, electrophoresis of
these macromolecules in solution results in little or no
separation of molecules of different lengths. H owever,
successful separation of proteins and nucleic acids can be
accomplished by electrophoresis in various gels (semisolid
suspensions in water) rather than in a liquid solution.
Electrophoretic separation of proteins is most commonly
performed in polyacrylam ide gels. When a mixture of
proteins is applied to a gel and an electric current is applied, smaller proteins migrate faster through the gel than
do larger proteins.
Gels are cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into polyacrylamide chains and simultaneously cross-linking the chains
into a semisolid matrix. The pore size of a gel can be varied
by adjusting the concentrations of polyacrylamide and the
cross-linking reagent. The rate at which a protein moves
through a gel is influenced by the gel’s pore size and the
strength of the electric field. By suitable adjustment of
these parameters, proteins of widely varying sizes can be
separated.
In the most powerful technique for resolving protein
mixtures, proteins are exposed to the ionic detergent SDS
(sodium dodecylsulfate) before and during gel electrophoresis (Figure 3-32). SDS denatures proteins, causing multimeric proteins to dissociate into their subunits, and
all polypeptide chains are forced into extended conformations with similar charge:mass ratios. SDS treatment thus
88
CHAPTER 3 • Protein Structure and Function
(a) Differential centrifugation
1 Sample is poured into tube
(b) Rate-zonal centrifugation
1 Sample is layered on top of gradient
Larger particle
M ore dense particle
Sm aller particle
Less dense particle
2
Centrifuge
Particles settle
according to
mass
Sucrose
gradient
2
Centrifugal
force
Centrifuge
Particles settle
according to
mass
Centrifugal force
3
Stop centrifuge
Decant liquid
into container
3
Stop centrifuge
Collect fractions
and do assay
Decreasing m ass of particles
▲ EX PERIM EN TA L FIGU RE 3 -3 1 Centrifugation techniques
separate particles that differ in mass or density. (a) In
differential centrifugation, a cell homogenate or other mixture is
spun long enough to sediment the denser particles (e.g., cell
organelles, cells), w hich collect as a pellet at the bottom of the
tube (step 2 ). The less dense particles (e.g., soluble proteins,
nucleic acids) remain in the liquid supernatant, w hich can be
eliminates the effect of differences in shape, and so chain
length, which corresponds to mass, is the sole determinant of
the migration rate of proteins in SDS-polyacrylamide electrophoresis. Even chains that differ in molecular weight by
less than 10 percent can be separated by this technique.
M oreover, the molecular weight of a protein can be estimated by comparing the distance that it migrates through a
gel with the distances that proteins of known molecular
weight migrate.
transferred to another tube (step 3 ). (b) In rate-zonal
centrifugation, a mixture is spun just long enough to separate
molecules that differ in mass but may be similar in shape and
density (e.g., globular proteins, RNA molecules) into discrete
zones w ithin a density gradient commonly formed by a
concentrated sucrose solution (step 2 ). Fractions are removed
from the bottom of the tube and assayed (step 5 ).
Two-Dimensional Gel Electrophoresis Electrophoresis of
all cellular proteins through an SDS gel can separate proteins
having relatively large differences in mass but cannot resolve
proteins having similar masses (e.g., a 41-kDa protein from
a 42-kDa protein). To separate proteins of similar masses,
another physical characteristic must be exploited. M ost commonly, this characteristic is electric charge, which is determined by the number of acidic and basic residues in a
protein. Two unrelated proteins having similar masses are
3.6 • Purifying, Detecting, and Characterizing Proteins
1
Denature sample w ith
sodium dodecylsulfate
Place mixture of proteins on gel,
apply electric field
_
Cross-linked
polyacrylam ide
gel
Partially
separated
proteins
Direction of m igration
+
3
Stain to visualize
separated bands
Decreasing
size
unlikely to have identical net charges because their sequences, and thus the number of acidic and basic residues,
are different.
In two-dimensional electrophoresis, proteins are separated sequentially, first by their charges and then by their
masses (Figure 3-33a). In the first step, a cell extract is
fully denatured by high concentrations (8 M ) of urea and
then layered on a gel strip that contains an continuous pH
gradient. The gradient is formed by am pholytes, a mixture
of polyanionic and polycationic molecules, that are cast
into the gel, with the most acidic ampholyte at one end
and the most basic ampholyte at the opposite end. A
charged protein will migrate through the gradient until it
reaches its isoelectric point (pI), the pH at which the net
charge of the protein is zero. This technique, called iso-
䉳 EX PERIM EN TA L FIGU RE 3 -3 2 SDSpolyacrylamide gel electrophoresis separates
proteins solely on the basis of their masses. Initial
treatment w ith SDS, a negatively charged detergent,
dissociates multimeric proteins and denatures all the
polypeptide chains (step 1 ). During electrophoresis,
the SDS-protein complexes migrate through the
polyacrylamide gel (step 2 ). Small proteins are able
to move through the pores more easily, and faster,
than larger proteins. Thus the proteins separate into
bands according to their sizes as they migrate through
the gel. The separated protein bands are visualized by
staining w ith a dye (step 3 ).
electric focusing (IEF), can resolve proteins that differ by
only one charge unit. Proteins that have been separated on
an IEF gel can then be separated in a second dimension on
the basis of their molecular weights. To accomplish this
separation, the IEF gel strip is placed lengthwise on a polyacrylamide slab gel, this time saturated with SDS. When an
electric field is imposed, the proteins will migrate from the
IEF gel into the SDS slab gel and then separate according
to their masses.
The sequential resolution of proteins by charge and mass
can achieve excellent separation of cellular proteins (Figure
3-33b). For example, two-dimensional gels have been very
useful in comparing the proteomes in undifferentiated and
differentiated cells or in normal and cancer cells because as
many as 1000 proteins can be resolved simultaneously.
M EDIA CON N ECTION S
Technique Animation: SDS Gel Electrophoresis
SDS-coated
proteins
2
89
CHAPTER 3 • Protein Structure and Function
Separate
in first
dimension
by charge
1
Isoelectric
focusing (IEF)
pH 10.0
Apply first gel
to top of second
pH 4.0
Isoelectric focusing ( 1 )
(b)
pH 4.0
2
pH 10.0
)
Protein
m ixture
3
66
SDS electrophoresis (
(a)
M olecular w eight ⫻ 10⫺3
90
43
30
16
Separate
in second
dimension
by size
3
SDS
electrophoresis
▲ EX PERIM EN TA L FIGU RE 3 -3 3 Two-dimensional gel
electrophoresis can separate proteins of similar mass. (a) In
this technique, proteins are first separated on the basis of their
charges by isoelectric focusing (step 1 ). The resulting gel strip is
applied to an SDS-polyacrylamide gel and the proteins are
separated into bands by mass (step 3 ). (b) In this two-
Liquid Chromatography Resolves Proteins
by Mass, Charge, or Binding Affinity
A third common technique for separating mixtures of proteins, as well as other molecules, is based on the principle
that molecules dissolved in a solution will interact (bind and
dissociate) with a solid surface. If the solution is allowed to
flow across the surface, then molecules that interact frequently with the surface will spend more time bound to the
surface and thus move more slowly than molecules that interact infrequently with the surface. In this technique, called
liquid chromatography, the sample is placed on top of a
tightly packed column of spherical beads held within a glass
cylinder. The nature of these beads determines whether the
separation of proteins depends on differences in mass,
charge, or binding affinity.
Gel Filtration Chromatography Proteins that differ in mass
can be separated on a column composed of porous beads
made from polyacrylamide, dextran (a bacterial polysaccharide), or agarose (a seaweed derivative), a technique called gel
filtration chromatography. Although proteins flow around the
spherical beads in gel filtration chromatography, they spend
some time within the large depressions that cover a bead’s surface. Because smaller proteins can penetrate into these depres-
4.2
5.9
pI
7.4
dimensional gel of a protein extract from cultured cells, each
spot represents a single polypeptide. Polypeptides can be
detected by dyes, as here, or by other techniques such as
autoradiography. Each polypeptide is characterized by its
isoelectric point (pI) and molecular weight. [Part (b) courtesy of
J. Celis.]
sions more easily than can larger proteins, they travel through
a gel filtration column more slowly than do larger proteins
(Figure 3-34a). (In contrast, proteins migrate through the
pores in an electrophoretic gel; thus smaller proteins move
faster than larger ones.) The total volume of liquid required
to elute a protein from a gel filtration column depends on its
mass: the smaller the mass, the greater the elution volume. By
use of proteins of known mass, the elution volume can be used
to estimate the mass of a protein in a mixture.
Ion-Exchange Chromatography In a second type of liquid
chromatography, called ion-ex change chrom atography, proteins are separated on the basis of differences in their
charges. This technique makes use of specially modified
beads whose surfaces are covered by amino groups or carboxyl groups and thus carry either a positive charge (N H 3 ⫹)
or a negative charge (CO O ⫺) at neutral pH .
The proteins in a mixture carry various net charges at
any given pH . When a solution of a protein mixture flows
through a column of positively charged beads, only proteins
with a net negative charge (acidic proteins) adhere to the
beads; neutral and positively charged (basic) proteins flow
unimpeded through the column (Figure 3-34b). The acidic
proteins are then eluted selectively by passing a gradient of
increasing concentrations of salt through the column. At low
91
3.6 • Purifying, Detecting, and Characterizing Proteins
(a) Gel filtration chrom atography
(c) Antibody-affinity chrom atography
Load in
pH 7 buffer
Large protein
Sm all protein
Layer
sample
on
column
Add buffer
to w ash
proteins
through
column
Polym er gel bead
Collect
fractions
3
2
1
Protein
recognized
by antibody
Elute
w ith
pH 3
buffer
Wash
Protein not
recognized
by antibody
Antibody
3
2
1
(b) Ion-exchange chrom atography
Negatively charged
protein
Positively charged
protein
Layer
sample
on
column
Collect
positively
charged
proteins
Elute negatively
charged protein
w ith salt solution
(NaCl)
Na+
Positively charged gel bead
▲ EX PERIM EN TA L FIGU RE 3 -3 4 Three commonly used
liquid chromatographic techniques separate proteins on the
basis of mass, charge, or affinity for a specific ligand. (a) Gel
filtration chromatography separates proteins that differ in size.
A mixture of proteins is carefully layered on the top of a glass
cylinder packed w ith porous beads. Smaller proteins travel
through the column more slow ly than larger proteins. Thus
different proteins have different elution volumes and can be
collected in separate liquid fractions from the bottom. (b) Ionexchange chromatography separates proteins that differ in net
charge in columns packed w ith special beads that carry either a
positive charge (show n here) or a negative charge. Proteins
salt concentrations, protein molecules and beads are attracted by their opposite charges. At higher salt concentrations, negative salt ions bind to the positively charged beads,
displacing the negatively charged proteins. In a gradient of
Cl −
4
3
2
1
having the same net charge as the beads are repelled and flow
through the column, w hereas proteins having the opposite
charge bind to the beads. Bound proteins—in this case,
negatively charged—are eluted by passing a salt gradient (usually
of NaCl or KCl) through the column. As the ions bind to the
beads, they desorb the protein. (c) In antibody-affinity
chromatography, a specific antibody is covalently attached to
beads packed in a column. Only protein w ith high affinity for the
antibody is retained by the column; all the nonbinding proteins
flow through. The bound protein is eluted w ith an acidic solution,
w hich disrupts the antigen–antibody complexes.
increasing salt concentration, weakly charged proteins are
eluted first and highly charged proteins are eluted last. Similarly, a negatively charged column can be used to retain and
fractionate basic proteins.
92
CHAPTER 3 • Protein Structure and Function
Affinity Chromatography The ability of proteins to bind
specifically to other molecules is the basis of affinity chrom atography. In this technique, ligand molecules that bind to
the protein of interest are covalently attached to the beads
used to form the column. Ligands can be enzyme substrates
or other small molecules that bind to specific proteins. In a
widely used form of this technique, antibody-affinity chrom atography, the attached ligand is an antibody specific for
the desired protein (Figure 3-34c).
An affinity column will retain only those proteins that
bind the ligand attached to the beads; the remaining proteins, regardless of their charges or masses, will pass
through the column without binding to it. H owever, if a retained protein interacts with other molecules, forming a
complex, then the entire complex is retained on the column.
The proteins bound to the affinity column are then eluted by
adding an excess of ligand or by changing the salt concentration or pH . The ability of this technique to separate particular proteins depends on the selection of appropriate
ligands.
Highly Specific Enzyme and Antibody Assays
Can Detect Individual Proteins
The purification of a protein, or any other molecule, requires
a specific assay that can detect the molecule of interest in column fractions or gel bands. An assay capitalizes on some
highly distinctive characteristic of a protein: the ability to
bind a particular ligand, to catalyze a particular reaction, or
to be recognized by a specific antibody. An assay must also
be simple and fast to minimize errors and the possibility that
the protein of interest becomes denatured or degraded while
the assay is performed. The goal of any purification scheme
is to isolate sufficient amounts of a given protein for study;
thus a useful assay must also be sensitive enough that only a
small proportion of the available material is consumed.
M any common protein assays require just from 10 ⫺9 to
10 ⫺12 g of material.
Chromogenic and Light-Emitting Enzyme Reactions M any
assays are tailored to detect some functional aspect of a protein. For example, enzyme assays are based on the ability to
detect the loss of substrate or the formation of product.
Some enzyme assays utilize chrom ogenic substrates, which
change color in the course of the reaction. (Some substrates
are naturally chromogenic; if they are not, they can be linked
to a chromogenic molecule.) Because of the specificity of an
enzyme for its substrate, only samples that contain the enzyme will change color in the presence of a chromogenic substrate and other required reaction components; the rate of
the reaction provides a measure of the quantity of enzyme
present.
Such chromogenic enzymes can also be fused or chemically linked to an antibody and used to “ report” the presence
or location of the antigen. Alternatively, luciferase, an enzyme present in fireflies and some bacteria, can be linked to
an antibody. In the presence of ATP and luciferin, luciferase
catalyzes a light-emitting reaction. In either case, after the
antibody binds to the protein of interest, substrates of the
linked enzyme are added and the appearance of color or
1 Electrophoresis/ transfer
Antibody detection
4 Chromogenic detection
2
3
Technique Animation: Immunoblotting
M EDIA CON N ECTION S
Electric
current
SDS-polyacrylam ide gel
M em brane
Incubate w ith
Ab 1 ( );
w ash excess
▲ EX PERIM EN TA L FIGU RE 3 -3 5 Western blotting
(immunoblotting) combines several techniques to resolve
and detect a specific protein. Step 1 : After a protein
mixture has been electrophoresed through an SDS gel, the
separated bands are transferred (blotted) from the gel onto a
porous membrane. Step 2 : The membrane is flooded w ith a
solution of antibody (Ab1) specific for the desired protein.
Only the band containing this protein binds the antibody,
forming a layer of antibody molecules (although their position
Incubate w ith enzym elinked Ab 2 ( );
w ash excess
React w ith substrate
for Ab 2-linked enzym e
cannot be seen at this point). After sufficient time for binding,
the membrane is washed to remove unbound Ab1. Step 3 :
The membrane is incubated w ith a second antibody (Ab2) that
binds to the bound Ab1. This second antibody is covalently
linked to alkaline phosphatase, w hich catalyzes a chromogenic
reaction. Step 4 : Finally, the substrate is added and a deep
purple precipitate forms, marking the band containing the
desired protein.
3.6 • Purifying, Detecting, and Characterizing Proteins
emitted light is monitored. A variation of this technique, particularly useful in detecting specific proteins within living
cells, makes use of green fluorescent protein (GFP), a naturally fluorescent protein found in jellyfish (see Figure 5-46).
Western Blotting A powerful method for detecting a particular protein in a complex mixture combines the superior
resolving power of gel electrophoresis, the specificity of antibodies, and the sensitivity of enzyme assays. Called Western
blotting, or immunoblotting, this multistep procedure is
commonly used to separate proteins and then identify a specific protein of interest. As shown in Figure 3-35, two different antibodies are used in this method, one specific for the
desired protein and the other linked to a reporter enzyme.
Radioisotopes Are Indispensable Tools
for Detecting Biological Molecules
A sensitive method for tracking a protein or other biological molecule is by detecting the radioactivity emitted from radioisotopes introduced into the molecule. At least one atom
in a radiolabeled molecule is present in a radioactive form,
called a radioisotope.
Radioisotopes Useful in Biological Research H undreds of
biological compounds (e.g., amino acids, nucleosides, and
numerous metabolic intermediates) labeled with various radioisotopes are commercially available. These preparations
vary considerably in their specific activity, which is the
amount of radioactivity per unit of material, measured in disintegrations per minute (dpm) per millimole. The specific activity of a labeled compound depends on the probability of
decay of the radioisotope, indicated by its half-life, which is
the time required for half the atoms to undergo radioactive
decay. In general, the shorter the half-life of a radioisotope,
the higher its specific activity (Table 3-3).
The specific activity of a labeled compound must be high
enough that sufficient radioactivity is incorporated into cellular molecules to be accurately detected. For example, methionine and cysteine labeled with sulfur-35 (35 S) are widely
used to label cellular proteins because preparations of these
`
TABLE 3-3
Radioisotopes Commonly Used
in Biological Research
Isotope
Half-Life
Phosphorus-32
14.3 days
Iodine-125
60.4 days
Sulfur-35
87.5 days
Tritium (hydrogen-3)
12.4 years
Carbon-14
5730.4 years
93
amino acids with high specific activities (>10 15 dpm/mmol)
are available. Likewise, commercial preparations of 3 H labeled nucleic acid precursors have much higher specific
activities than those of the corresponding 14 C-labeled preparations. In most experiments, the former are preferable because they allow RN A or DN A to be adequately labeled after
a shorter time of incorporation or require a smaller cell sample. Various phosphate-containing compounds in which
every phosphorus atom is the radioisotope phosphorus-32
are readily available. Because of their high specific activity,
32
P-labeled nucleotides are routinely used to label nucleic
acids in cell-free systems.
Labeled compounds in which a radioisotope replaces
atoms normally present in the molecule have the same chemical properties as the corresponding nonlabeled compounds.
Enzymes, for instance, cannot distinguish between substrates
labeled in this way and their nonlabeled substrates. In contrast, labeling with the radioisotope iodine-125 (125 I) requires the covalent addition of 125 I to a protein or nucleic
acid. Because this labeling procedure modifies the chemical
structure of a protein or nucleic acid, the biological activity
of the labeled molecule may differ somewhat from that of the
nonlabeled form.
Labeling Experiments and Detection of Radiolabeled
Molecules Whether labeled compounds are detected by autoradiography, a semiquantitative visual assay, or their radioactivity is measured in an appropriate “ counter,” a highly
quantitative assay that can determine the concentration of a
radiolabeled compound in a sample, depends on the nature
of the experiment. In some experiments, both types of detection are used.
In one use of autoradiography, a cell or cell constituent
is labeled with a radioactive compound and then overlaid
with a photographic emulsion sensitive to radiation. Development of the emulsion yields small silver grains whose distribution corresponds to that of the radioactive material.
Autoradiographic studies of whole cells were crucial in determining the intracellular sites where various macromolecules are synthesized and the subsequent movements of these
macromolecules within cells. Various techniques employing
fluorescent microscopy, which we describe in the next chapter, have largely supplanted autoradiography for studies of
this type. H owever, autoradiography is commonly used in
various assays for detecting specific isolated DN A or RN A
sequences (Chapter 9).
Q uantitative measurements of the amount of radioactivity in a labeled material are performed with several different
instruments. A G eiger counter measures ions produced in a
gas by the  particles or ␥ rays emitted from a radioisotope.
In a scintillation counter, a radiolabeled sample is mixed with
a liquid containing a fluorescent compound that emits a flash
of light when it absorbs the energy of the  particles or ␥ rays
released in the decay of the radioisotope; a phototube in the
instrument detects and counts these light flashes. Phosphorim agers are used to detect radiolabeled compounds on a surface, storing digital data on the number of decays in
CHAPTER 3 • Protein Structure and Function
ER
Golgi
Secretory
granule
Pulse
T = 0;
add 3H-leucine
Chase
T = 5 m in;
w ash out 3H-leucine
T = 10 m in
T = 45 m in
▲ EX PERIM EN TA L FIGU RE 3 -3 6 Pulse-chase experiments
can track the pathway of protein movement w ithin cells.
To determine the pathway traversed by secreted proteins
subsequent to their synthesis on the rough endoplasmic
reticulum (ER), cells are briefly incubated in a medium containing
a radiolabeled amino acid (e.g., [3H]leucine), the pulse, w hich w ill
label any protein synthesized during this period. The cells are
then washed w ith buffer to remove the pulse and transferred to
medium lacking a radioactive precursor, the chase. Samples
taken periodically are analyzed by autoradiography to determine
the cellular location of labeled protein. At the beginning of the
experiment (t ⫽ 0), no protein is labeled, as indicated by the
green dotted lines. At the end of the pulse (t ⫽ 5 minutes), all
the labeled protein (red lines) appears in the ER. At subsequent
times, this new ly synthesized labeled protein is visualized first
in the Golgi complex and then in secretory vesicles. Because
any protein synthesized during the chase period is not labeled,
the movement of the labeled protein can be defined quite
precisely.
disintegrations per minute per small pixel of surface area.
These instruments, which can be thought of as a kind of
reusable electronic film, are commonly used to quantitate radioactive molecules separated by gel electrophoresis and are
replacing photographic emulsions for this purpose.
A combination of labeling and biochemical techniques
and of visual and quantitative detection methods is often employed in labeling experiments. For instance, to identify the
major proteins synthesized by a particular cell type, a sample
of the cells is incubated with a radioactive amino acid (e.g.,
[35 S]methionine) for a few minutes. The mixture of cellular
proteins is then resolved by gel electrophoresis, and the gel
is subjected to autoradiography or phosphorimager analysis.
The radioactive bands correspond to newly synthesized proteins, which have incorporated the radiolabeled amino acid.
Alternatively, the proteins can be resolved by liquid chromatography, and the radioactivity in the eluted fractions can
be determined quantitatively with a counter.
Pulse-chase experiments are particularly useful for tracing changes in the intracellular location of proteins or the
transformation of a metabolite into others over time. In this
experimental protocol, a cell sample is exposed to a radiolabeled compound—the “ pulse” —for a brief period of time,
then washed with buffer to remove the labeled pulse, and finally incubated with a nonlabeled form of the compound—
the “ chase” (Figure 3-36). Samples taken periodically are
assayed to determine the location or chemical form of the
radiolabel. A classic use of the pulse-chase technique was in
studies to elucidate the pathway traversed by secreted proteins from their site of synthesis in the endoplasmic reticulum
to the cell surface (Chapter 17).
Mass Spectrometry Measures the Mass
of Proteins and Peptides
A powerful technique for measuring the mass of molecules
such as proteins and peptides is m ass spectrom etry. This
Laser
M etal
target
1 Ionization
+
+
2 Acceleration
Sam ple
Intensity
94
+
3
Detection
Lightest ions
arrive at
detector first
Tim e
▲ EX PERIM EN TA L FIGU RE 3 -3 7 The molecular weight of
proteins and peptides can be determined by time-of-flight
mass spectrometry. In a laser-desorption mass spectrometer,
pulses of light from a laser ionize a protein or peptide mixture
that is absorbed on a metal target ( 1 ). An electric field
accelerates the molecules in the sample toward the detector
( 2 and 3 ). The time to the detector is inversely proportional
to the mass of a molecule. For molecules having the same
charge, the time to the detector is inversely proportional to the
mass. The molecular weight is calculated using the time of flight
of a standard.
3.6 • Purifying, Detecting, and Characterizing Proteins
technique requires a method for ionizing the sample, usually
a mixture of peptides or proteins, accelerating the molecular ions, and then detecting the ions. In a laser desorption
mass spectrometer, the protein sample is mixed with an organic acid and then dried on a metal target. Energy from a
laser ionizes the proteins, and an electric field accelerates the
ions down a tube to a detector (Figure 3-37). Alternatively, in
an electrospray mass spectrometer, a fine mist containing the
sample is ionized and then introduced into a separation
chamber where the positively charged molecules are accelerated by an electric field. In both instruments, the time of
flight is inversely proportional to a protein’s mass and directly proportional to its charge. As little as 1 ⫻ 10 ⫺15 mol
(1 femtomole) of a protein as large as 200,000 M W can be
measured with an error of 0.1 percent.
Protein Primary Structure Can Be Determined
by Chemical Methods and from Gene Sequences
The classic method for determining the amino acid sequence
of a protein is Edm an degradation. In this procedure, the free
amino group of the N -terminal amino acid of a polypeptide
is labeled, and the labeled amino acid is then cleaved from
the polypeptide and identified by high-pressure liquid chromatography. The polypeptide is left one residue shorter, with
a new amino acid at the N -terminus. The cycle is repeated on
the ever shortening polypeptide until all the residues have
been identified.
Before about 1985, biologists commonly used the Edman
chemical procedure for determining protein sequences. N ow,
however, protein sequences are determined primarily by
analysis of genome sequences. The complete genomes of several organisms have already been sequenced, and the database of genome sequences from humans and numerous
model organisms is expanding rapidly. As discussed in Chapter 9, the sequences of proteins can be deduced from DN A
sequences that are predicted to encode proteins.
A powerful approach for determining the primary structure of an isolated protein combines mass spectroscopy and
the use of sequence databases. First, mass spectrometry is
used to determine the peptide m ass fingerprint of the protein.
A peptide mass fingerprint is a compilation of the molecular
weights of peptides that are generated by a specific protease.
The molecular weights of the parent protein and its proteolytic fragments are then used to search genome databases
for any similarly sized protein with identical or similar peptide mass maps.
Peptides with a Defined Sequence Can Be
Synthesized Chemically
Synthetic peptides that are identical with peptides synthesized in vivo are useful experimental tools in studies of proteins and cells. For example, short synthetic peptides of
10–15 residues can function as antigens to trigger the production of antibodies in animals. A synthetic peptide, when
95
coupled to a large protein carrier, can trick an animal into
producing antibodies that bind the full-sized, natural protein
antigen. As we’ll see throughout this book, antibodies are extremely versatile reagents for isolating proteins from mixtures by affinity chromatography (see Figure 3-34c), for
separating and detecting proteins by Western blotting (see
Figure 3-35), and for localizing proteins in cells by microscopic techniques described in Chapter 5.
Peptides are routinely synthesized in a test tube from
monomeric amino acids by condensation reactions that form
peptide bonds. Peptides are constructed sequentially by coupling the C-terminus of a monomeric amino acid with the N terminus of the growing peptide. To prevent unwanted
reactions entailing the amino groups and carboxyl groups
of the side chains during the coupling steps, a protecting
(blocking) group is attached to the side chains. Without these
protecting groups, branched peptides would be generated. In
the last steps of synthesis, the side chain–protecting groups
are removed and the peptide is cleaved from the resin on
which synthesis takes place.
Protein Conformation Is Determined
by Sophisticated Physical Methods
In this chapter, we have emphasized that protein function is
dependent on protein structure. Thus, to figure out how a
protein works, its three-dimensional structure must be
known. Determining a protein’s conformation requires sophisticated physical methods and complex analyses of the experimental data. We briefly describe three methods used to
generate three-dimensional models of proteins.
X-Ray Crystallography The use of x-ray crystallography to
determine the three-dimensional structures of proteins was
pioneered by M ax Perutz and John Kendrew in the 1950s. In
this technique, beams of x-rays are passed through a protein
crystal in which millions of protein molecules are precisely
aligned with one another in a rigid array characteristic of the
protein. The wavelengths of x-rays are about 0.1–0.2 nm,
short enough to resolve the atoms in the protein crystal.
Atoms in the crystal scatter the x-rays, which produce a diffraction pattern of discrete spots when they are intercepted
by photographic film (Figure 3-38). Such patterns are extremely complex—composed of as many as 25,000 diffraction spots for a small protein. Elaborate calculations and
modifications of the protein (such as the binding of heavy
metals) must be made to interpret the diffraction pattern and
to solve the structure of the protein. The process is analogous
to reconstructing the precise shape of a rock from the ripples that it creates in a pond. To date, the detailed threedimensional structures of more than 10,000 proteins have
been established by x-ray crystallography.
Cryoelectron Microscopy Although some proteins readily
crystallize, obtaining crystals of others—particularly large
multisubunit proteins—requires a time-consuming trial-and-
96
CHAPTER 3 • Protein Structure and Function
(a)
of electrons to prevent radiation-induced damage to the structure. Sophisticated computer programs analyze the images
and reconstruct the protein’s structure in three dimensions.
Recent advances in cryoelectron microscopy permit researchers to generate molecular models that compare with
those derived from x-ray crystallography. The use of cryoelectron microscopy and other types of electron microscopy
for visualizing cell structures are discussed in Chapter 5.
X-ray
source
X-ray
beam
Crystal
Detector
(e.g., film )
Diffracted
beam s
NMR Spectroscopy The three-dimensional structures of
small proteins containing about as many as 200 amino acids
can be studied with nuclear magnetic resonance (N M R)
spectroscopy. In this technique, a concentrated protein solution is placed in a magnetic field and the effects of different
radio frequencies on the spin of different atoms are measured. The behavior of any atom is influenced by neighboring atoms in adjacent residues, with closely spaced residues
being more perturbed than distant residues. From the magnitude of the effect, the distances between residues can be
calculated; these distances are then used to generate a model
of the three-dimensional structure of the protein.
Although N M R does not require the crystallization of a
protein, a definite advantage, this technique is limited to proteins smaller than about 20 kDa. H owever, N M R analysis
can also be applied to protein domains, which tend to be
small enough for this technique and can often be obtained
as stable structures.
K EY C O N C EP T S O F S EC T I O N 3 . 6
Purifying, Detecting, and Characterizing Proteins
Proteins can be separated from other cell components
and from one another on the basis of differences in their
physical and chemical properties.
■
Centrifugation separates proteins on the basis of their
rates of sedimentation, which are influenced by their
masses and shapes.
■
▲ EX PERIM EN TA L FIGU RE 3 -3 8 X-ray crystallography
provides diffraction data from w hich the three-dimensional
structure of a protein can be determined. (a) Basic
components of an x-ray crystallographic determination. When a
narrow beam of x-rays strikes a crystal, part of it passes straight
through and the rest is scattered (diffracted) in various directions.
The intensity of the diffracted waves is recorded on an x-ray film
or w ith a solid-state electronic detector. (b) X-ray diffraction
pattern for a topoisomerase crystal collected on a solid-state
detector. From complex analyses of patterns like this one, the
location of every atom in a protein can be determined. [Part (a)
adapted from L. Stryer, 1995, Biochemistry, 4th ed., W. H. Freeman and
Company, p. 64; part (b) courtesy of J. Berger.]
Gel electrophoresis separates proteins on the basis of
their rates of movement in an applied electric field. SDSpolyacrylamide gel electrophoresis can resolve polypeptide
chains differing in molecular weight by 10 percent or less
(see Figure 3-32).
■
Liquid chromatography separates proteins on the basis
of their rates of movement through a column packed with
spherical beads. Proteins differing in mass are resolved on
gel filtration columns; those differing in charge, on ionexchange columns; and those differing in ligand-binding
properties, on affinity columns (see Figure 3-34).
■
Various assays are used to detect and quantify proteins.
Some assays use a light-producing reaction or radioactivity to generate a signal. O ther assays produce an amplified
colored signal with enzymes and chromogenic substrates.
■
error effort to find just the right conditions. The structures
of such difficult-to-crystallize proteins can be obtained by cryoelectron m icroscopy. In this technique, a protein sample is
rapidly frozen in liquid helium to preserve its structure and
then examined in the frozen, hydrated state in a cryoelectron
microscope. Pictures are recorded on film by using a low dose
Antibodies are powerful reagents used to detect, quantify, and isolate proteins. They are used in affinity chromatography and combined with gel electrophoresis in
■
Review the Concepts
Western blotting, a powerful method for separating and
detecting a protein in a mixture (see Figure 3-35).
■ Autoradiography is a semiquantitative technique for detecting radioactively labeled molecules in cells, tissues, or
electrophoretic gels.
■ Pulse-chase labeling can determine the intracellular fate
of proteins and other metabolites (see Figure 3-36).
■ Three-dimensional structures of proteins are obtained by
x-ray crystallography, cryoelectron microscopy, and N M R
spectroscopy. X-ray crystallography provides the most detailed structures but requires protein crystallization. Cryoelectron microscopy is most useful for large protein complexes, which are difficult to crystallize. O nly relatively
small proteins are amenable to N M R analysis.
PERS PECT I V ES FO R T H E FU T U RE
Impressive expansion of the computational power of computers is at the core of advances in determining the threedimensional structures of proteins. For example, vacuum
tube computers running on programs punched on cards
were used to solve the first protein structures on the basis
of x-ray crystallography. In the future, researchers aim to
predict the structures of proteins only on the basis of
amino acid sequences deduced from gene sequences. This
computationally challenging problem requires supercomputers or large clusters of computers working in synchrony. Currently, only the structures of very small
domains containing 100 residues or fewer can be predicted
at a low resolution. H owever, continued developments in
computing and models of protein folding, combined with
large-scale efforts to solve the structures of all protein motifs by x-ray crystallography, will allow the prediction of
the structures of larger proteins. With an exponentially expanding database of motifs, domains, and proteins, scientists will be able to identify the motifs in an unknown
protein, match the motif to the sequence, and use this head
start in predicting the three-dimensional structure of the
entire protein.
N ew combined approaches will also help in in determining high-resolution structures of molecular machines such as
those listed in Table 3-1. Although these very large macromolecular assemblies usually are difficult to crystallize and
thus to solve by x-ray crystallography, they can be imaged in
a cryoelectron microscope at liquid helium temperatures and
high electron energies. From millions of individual “ particles,” each representing a random view of the protein complex, the three-dimensional structure can be built. Because
subunits of the complex may already be solved by crystallography, a composite structure consisting of the x-ray-derived
subunit structures fitted to the EM -derived model will be generated. An interesting application of this type of study would
be the solution of the structures of amyloid and prion pro-
97
teins, especially in the early stages in the formation of insoluble filaments.
Understanding the operation of protein machines will require the measurement of many new characteristics of proteins. For example, because many machines do nonchemical
work of some type, biologists will have to identify the energy sources (mechanical, electrical, or thermal) and measure the amounts of energy to determine the limits of a
particular machine. Because most activities of machines include movement of one type or another, the force powering
the movement and its relation to biological activity can be
a source of insight into how force generation is coupled to
chemistry. Improved tools such as optical traps and atomic
force microscopes will enable detailed studies of the forces
and chemistry pertinent to the operation of individual protein machines.
KEY T ERM S
␣ helix 61
activation energy 74
active site 75
allostery 83
amyloid filament 73
autoradiography 93
 sheet 61
chaperone 69
conformation 60
cooperativity 83
domain 63
electrophoresis 87
homology 68
K m 76
ligand 73
liquid chromatography 90
molecular machine 59
motif 63
motor protein 79
peptide bond 60
polypeptide 61
primary structure 61
proteasome 71
protein 61
proteome 60
quaternary structure 66
rate-zonal centrifugation 87
secondary structure 61
tertiary structure 62
ubiquitin 71
V max 76
x-ray crystallography 95
REV I EW T H E CO N CEPT S
1. The three-dimensional structure of a protein is determined by its primary, secondary, and tertiary structures.
Define the primary, secondary, and tertiary structures. What
are some of the common secondary structures? What are the
forces that hold together the secondary and tertiary structures? What is the quaternary structure?
2. Proper folding of proteins is essential for biological
activity. Describe the roles of molecular chaperones and
chaperonins in the folding of proteins.
3. Proteins are degraded in cells. What is ubiquitin, and
what role does it play in tagging proteins for degradation?
What is the role of proteasomes in protein degradation?
98
CHAPTER 3 • Protein Structure and Function
4. Enzymes can catalyze chemical reactions. H ow do enzymes increase the rate of a reaction? What constitutes the
active site of an enzyme? For an enzyme-catalyzed reaction,
what are K m and V max ? For enzyme X, the K m for substrate
A is 0.4 mM and for substrate B is 0.01 mM . Which substrate has a higher affinity for enzyme X?
5. M otor proteins, such as myosin, convert energy into a
mechanical force. Describe the three general properties characteristic of motor proteins. Describe the biochemical events
that occur during one cycle of movement of myosin relative
to an actin filament.
6. The function of proteins can be regulated in a number of
ways. What is cooperativity, and how does it influence protein function? Describe how protein phosphorylation and
proteolytic cleavage can modulate protein function.
7. A number of techniques can separate proteins on the
basis of their differences in mass. Describe the use of two of
these techniques, centrifugation and gel electrophoresis. The
blood proteins transferrin (M W 76 kDa) and lysozyme (M W
15 kDa) can be separated by rate zonal centrifugation or SDS
polyacrylamide gel electrophoresis. Which of the two proteins will sediment faster during centrifugation? Which will
migrate faster during electrophoresis?
shown below. What do you conclude about the effect of the
drug on the steady-state levels of proteins 1–7?
Control
pH
4
−
1
10
4
+ Drug
pH
10
−
2
3
4
6
5
7
+
+
b. You suspect that the drug may be inducing a protein
kinase and so repeat the experiment in part a in the presence
of 32 P-labeled inorganic phosphate. In this experiment the
two-dimensional gels are exposed to x-ray film to detect the
presence of 32 P-labeled proteins. The x-ray films are shown
below. What do you conclude from this experiment about
the effect of the drug on proteins 1–7?
4
Control
pH
10
4
−
−
+
+
+ Drug
pH
10
8. Chromatography is an analytical method used to separate proteins. Describe the principles for separating proteins
by gel filtration, ion-exchange, and affinity chromatography.
9. Various methods have been developed for detecting
proteins. Describe how radioisotopes and autoradiography
can be used for labeling and detecting proteins. H ow does
Western blotting detect proteins?
10. Physical methods are often used to determine protein
conformation. Describe how x-ray crystallography, cryoelectron microscopy, and N M R spectroscopy can be used to
determine the shape of proteins.
c. To determine the cellular localization of proteins 1–7, the
cells from part a were separated into nuclear and cytoplasmic
fractions by differential centrifugation. Two-dimensional gels
were run and the stained gels are shown below. What do you
conclude about the cellular localization of proteins 1–7?
Control
4
A N A LY Z E T H E DATA
Proteomics involves the global analysis of protein expression. In one approach, all the proteins in control cells and
treated cells are extracted and subsequently separated using
two-dimensional gel electrophoresis. Typically, hundreds or
thousands of protein spots are resolved and the steady-state
levels of each protein are compared between control and
treated cells. In the following example, only a few protein
spots are shown for simplicity. Proteins are separated in the
first dimension on the basis of charge by isoelectric focusing
(pH 4–10) and then separated by size by SDS polyacrylamide
gel electrophoresis. Proteins are detected with a stain such
as Coomassie blue and assigned numbers for identification.
a. Cells are treated with a drug (“ ⫹ Drug” ) or left untreated
(“ Control” ) and then proteins are extracted and separated
by two-dimensional gel electrophoresis. The stained gels are
Nuclear
pH
10
4
−
−
+
+
Cytoplasm ic
pH
10
+ Drug
4
Nuclear
pH
10
4
−
−
+
+
Cytoplasm ic
pH
10
References
d. Summarize the overall properties of proteins 1–7, combining the data from parts a, b, and c. Describe how you
could determine the identity of any one of the proteins.
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