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Protein Structure

Mary K. Campbell • Many conformations are possible for proteins:


Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
• Due to flexibility of amino acids linked by peptide
bonds

Chapter Four • At least one major conformations has biological


The Three-Dimensional Structure of Proteins activity, and hence is considered the protein’s native
conformation

Paul D. Adams • University of Arkansas

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Levels of Protein Structure 1˚ Structure


1° structure: the sequence of amino acids in a • The 1˚ sequence of proteins determines its 3-D
polypeptide chain, read from the N-terminal end to conformation
the C-terminal end
• 2° structure: the ordered 3-dimensional • Changes in just one amino acid in sequence can alter
arrangements (conformations) in localized regions of biological function, e.g. hemoglobin associated with
a polypeptide chain; refers only to interactions of the
peptide backbone sickle-cell anemia
• e. g., -helix and -pleated sheet
• 3˚ structure: 3-D arrangement of all atoms • Determination of 1˚ sequence is routine biochemistry
• 4˚ structure: arrangement of monomer subunits with lab work (See Ch. 5).
respect to each other

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2˚ Structure -Helix
• 2˚ of proteins is hydrogen-bonded arrangement of • Coil of the helix is clockwise or right-handed
backbone of the protein • There are 3.6 amino acids per turn
• Two bonds have free rotation: • Repeat distance is 5.4Å
• Each peptide bond is s-trans and planar
1) Bond between -carbon and amino nitrogen in
• C=O of each peptide bond is hydrogen bonded to the
residue N-H of the fourth amino acid away
2) Bond between the -carbon and carboxyl carbon of • C=O----H-N hydrogen bonds are parallel to helical
residue axis
• See Figure 4.1 • All R groups point outward from helix

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1
-Helix (Cont’d) -Helix (Cont’d)
• Several factors can disrupt an -helix
• proline creates a bend because of (1) the restricted
rotation due to its cyclic structure and (2) its -amino
group has no N-H for hydrogen bonding
• strong electrostatic repulsion caused by the proximity
of several side chains of like charge, e.g., Lys and Arg
or Glu and Asp
• steric crowding caused by the proximity of bulky side
chains, e.g., Val, Ile, Thr

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-Pleated Sheet -Pleated Sheet (Cont’d)


• Polypeptide chains lie adjacent to one another; may
be parallel or antiparallel
• R groups alternate, first above and then below plane
• Each peptide bond is s-trans and planar
• C=O and N-H groups of each peptide bond are
perpendicular to axis of the sheet
• C=O---H-N hydrogen bonds are between adjacent
sheets and perpendicular to the direction of the sheet

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-Pleated Sheet (Cont’d) Structures of Reverse Turns

-bulge- a common nonrepetive irregular 2˚ motif in • Glycine found in reverse turns


anti-parallel structure • Spatial (steric) reasons
• Polypeptide changes direction
• Proline also encountered in reverse turns. Why?

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2
Schematic Diagrams of Supersecondary
-Helices and -Sheets
Structures
• Supersecondary structures: the combination of -
and -sections, as for example
•  unit: two parallel strands of -sheet connected by
a stretch of -helix
•  unit: two antiparallel -helices
• -meander: an antiparallel sheet formed by a series of
tight reverse turns connecting stretches of a
polypeptide chain
• Greek key: a repetitive supersecondary structure
formed when an antiparallel sheet doubles back on
itself
• -barrel: created when -sheets are extensive enough
to fold back on themselves

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Collagen Triple Helix Fibrous Proteins


• Consists of three polypeptide chains wrapped around • Fibrous proteins: contain polypeptide chains
each other in a ropelike twist to form a triple helix organized approximately parallel along a single axis.
called tropocollagen; MW approx. 300,000 They
• consist of long fibers or large sheets
• 30% of amino acids in each chain are Pro and Hyp
• tend to be mechanically strong
(hydroxyproline); hydroxylysine also occurs
• are insoluble in water and dilute salt solutions
• Every third position is Gly and repeating sequences • play important structural roles in nature
are X-Pro-Gly and X-Hyp-Gly
• Examples are
• Each polypeptide chain is a helix but not an -helix • keratin of hair and wool
• The three strands are held together by hydrogen • collagen of connective tissue of animals including
bonding involving hydroxyproline and hydroxylysine cartilage, bones, teeth, skin, and blood vessels
• With age, collagen helices become cross linked by
covalent bonds formed between Lys and His residues

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Comparison of Shapes of Fibrous and


Globular Proteins
Globular Proteins
• Globular proteins: proteins which are folded to a
more or less spherical shape
• they tend to be soluble in water and salt solutions
• most of their polar side chains are on the outside and
interact with the aqueous environment by hydrogen
bonding and ion-dipole interactions
• most of their nonpolar side chains are buried inside
• nearly all have substantial sections of -helix and -
sheet

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3
3˚ Structure Forces in 3˚ Structure
• The 3-dimensional arrangement of atoms in the • Noncovalent interactions, including
molecule. • hydrogen bonding between polar side chains, e.g., Ser
and Thr
• In fibrous protein, backbone of protein does not fall • hydrophobic interaction between nonpolar side chains,
back on itself, it is important aspect of 3˚ not specified e.g., Val and Ile
by 2˚ structure. • electrostatic attraction between side chains of opposite
charge, e.g., Lys and Glu
• electrostatic repulsion between side chains of like
• In globular protein, more information needed. 3k charge, e.g., Lys and Arg, Glu and Asp
structure allows for the determination of the way
helical and pleated-sheet sections fold back on each • Covalent interactions: Disulfide (-S-S-) bonds
other. between side chains of cysteines

• Interactions between side chains also plays a role.

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Forces That Stabilize Protein Structure 3° and 4° Structure


• Tertiary (3°) structure: the arrangement in space of
all atoms in a polypeptide chain
• it is not always possible to draw a clear distinction
between 2° and 3° structure

• Quaternary (4°) structure: the association of


polypeptide chains into aggregations
• Proteins are divided into two large classes based on
their three-dimensional structure
• fibrous proteins
• globular proteins

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Determination of 3° Structure X-Ray and NMR Data


• X-ray crystallography
• uses a perfect crystal; that is, one in which all
individual protein molecules have the same 3D
structure and orientation
• exposure to a beam of x-rays gives a series diffraction
patterns
• information on molecular coordinates is extracted by a
mathematical analysis called a Fourier series
• 2-D Nuclear magnetic resonance High resolution method to determine 3˚
structure of proteins (from crystal)
• can be done on protein samples in aqueous solution Determines solution structure
Diffraction pattern produced by electrons
Structural info. Gained from
scattering X-rays
determining distances between
Series of patterns taken at different nuclei that aid in structure
angles gives structural information determination

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4
Myoglobin The Structure of Myoglobin
• A single polypeptide chain of 153 amino acids
• A single heme group in a hydrophobic pocket
• 8 regions of -helix; no regions of -sheet
• Most polar side chains are on the surface
• Nonpolar side chains are folded to the interior
• Two His side chains are in the interior, involved with
interaction with the heme group
• Fe(II) of heme has 6 coordinates sites; 4 interact with
N atoms of heme, 1 with N of a His side chain, and 1
with either an O2 molecule or an N of the second His
side chain

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Oxygen Binding Site of Myoglobin Denaturation


• Denaturation: the loss of the structural order (2°, 3°,
4°, or a combination of these) that gives a protein its
biological activity; that is, the loss of biological activity

• Denaturation can be brought about by


• heat
• large changes in pH, which alter charges on side
chains, e.g., -COO- to -COOH or -NH+ to -NH
• detergents such as sodium dodecyl sulfate (SDS)
which disrupt hydrophobic interactions
• urea or guanidine, which disrupt hydrogen bonding
• mercaptoethanol, which reduces disulfide bonds

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Denaturation and Refolding in


Denaturation of a Protein
Ribonuclease

Several ways to denature


proteins
• Heat
• pH
• Detergents
• Urea
• Guanadine hydrochloride

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5
Quaternary Structure Oxygen Binding of Hemoglobin (Hb)
• Quaternary (4°) structure: the association of • A tetramer of two -chains (141 amino acids each)
polypepetide monomers into multisubunit proteins and two -chains (153 amino acids each); 22
• dimers • Each chain has 1 heme group; hemoglobin can bind
• trimers up to 4 molecules of O2
• tetramers • Binding of O2 exhibited by positive cooperativity;
when one O2 is bound, it becomes easier for the next
• Noncovalent interactions O2 to bind
• electrostatics, hydrogen bonds, hydrophobic • The function of hemoglobin is to transport oxygen
• The structure of oxygenated Hb is different from that
of unoxygenated Hb
• H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG)
affect the ability of Hb to bind and transport oxygen

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Structure of Hemoglobin Conformation Changes That Accompany Hb Function

• Structural changes occur during binding of small


molecules
• Characteristic of allosteric behavior
• Hb exhibits different 4˚ structure in the bound and
unbound oxygenated forms
• Other ligands are involved in cooperative effect of Hb
can affect protein’s affinity for O 2 by altering structure

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Oxy- and Deoxyhemoglobin Protein Folding Dynamics


• Can 3˚ structure of protein be predicted? Yes, within
limitations
• The integration of biochemistry and computing has led
to bioinformatics
• Protein structure prediction is one of the principal
application of bioinformatics
• First step to predict protein structure is to search for
sequence homology

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6
Predicting Protein Structure Hydrophobic Interactions
• Hydrophobic interactions are major factors in protein
folding
• Folds so that nonpolar hydrophobic side chains tend
to be on inside away from water, and polar side chains
on outside accessible to aqueous environment
• Hydrophobic interactions are spontaneous

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Hydrophobic and Hydrophilic Interactions in Proteins Protein Folding Chaperones


• In the protein-dense environment of a cell, proteins
may begin to fold incorrectly or may associate with
other proteins before folding is completed

• Special proteins called chaperones aid in the correct


and timely folding of many proteins

• hsp70 were the first chaperone proteins discovered

• Chaperones exist in organisms from prokaryotes to


humans

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