Chapter 3: Amino Acids, Peptides,

and Proteins
Dr. Clower
Chem 4202
 Outline (part I)

 Sections 3.1 and 3.2

 Amino Acids
 Chemical structure
 Acid-base properties
 Stereochemistry
 Non-standard amino acids
 Formation of Peptide Bonds
 Amino Acids

 The building blocks of proteins

 Also used as single molecules in biochemical
pathways
 20 standard amino acids (-amino acids)
 Two functional groups:
 carboxylic acid group
 amino group on the alpha () carbon
 Have different side groups (R) R side chain
|
 Properties dictate behavior of AAs H2N— C —COOH
|
H
 Zwitterions

 Both the –NH2 and the –COOH groups in an amino acid

undergo ionization in water.
 At physiological pH (7.4), a zwitterion forms
 Both + and – charges
 Overall neutral
 Amphoteric
 Amino group is protonated
 Carboxyl group is deprotonated

 Soluble in polar solvents due to ionic character

 Structure of R also influence solubility
 Classification of Amino Acids

 Classify by structure of R
 Nonpolar
 Polar
 Aromatic
 Acidic
 Basic
 Nonpolar Amino Acids

 Hydrophobic, neutral, aliphatic

 Polar Amino Acids

 Hydrophilic, neutral, typically H-bond

 Disulfide Bonds

 Formed from oxidation of cysteine residues

 Aromatic Amino Acids

 Bulky, neutral, polarity depend on R

 Acidic and Basic Amino Acids

 Acidic  Basic
 R group = carboxylic  R group = amine
acid  Accepts H+
 Donates H+  Positively charged
 Negatively charged  His ionizes at pH 6.0
 Acid-base Properties

 Remember H3PO4 (multiple pKa’s)

 AAs also have multiple pKa’s due to multiple ionizable
groups

pK1 ~ 2.2
(protonated below 2.2)

pK2 ~ 9.4
(NH3+ below 9.4)

pKR
(when applicable)
 Table 3-1

Note 3-letter
and 1-letter
abbreviations

Amino acid organ

ization chart
 pH and Ionization

 Consider glycine:

O O O

OH- OH-
H3N CH C OH H3N CH C O H2N CH C O
+ +
H3O H3O
H H H

Glycine ion at Zwitterion of glycine Glycine ion at

acidic pH (charge = 0) basic pH
(charge = 1+) (charge = 1-)

 Note that the uncharged species never forms

 Titration of Glycine

 pK1
 [cation] = [zwitterion]
 pK2
 [zwitterion] = [anion]
 First equivalence point
 Zwitterion
 Molecule has no net charge
 pH = pI (Isoelectric point)
 pI = average of pKa’s = ½ (pK1 + pK2)
 pIglycine = ½ (2.34 + 9.60) = 5.97
 Animation
 pI of Lysine

 For AAs with 3 pKa’s, pI = average of two relevant pKa values

 Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53):
O O O O
pK1 pK2 pKR
H3N CH C OH H3N CH C O H2N CH C O H2N CH C O

CH2CH2CH2CH2NH3+ CH2CH2CH2CH2NH3+ CH2CH2CH2CH2NH3+ CH2CH2CH2CH2NH2

 Which species is the isoelectric form?

 So, pI = ½ (pK2 + pKR)
= ½ (8.95 + 10.53) = 9.74

 Note: pKR is not always higher than pK2 (see Table 3-1 and Fig. 3-12)
 Learning Check

 Would the following ions of serine exist at a

pH above, below, or at pI?
O O O

H3N CH C O H3N CH C OH H2N CH C O

CH2 CH2 CH2

OH OH OH
 Stereochemistry of AAs

 All amino acids (except glycine) are optically active

 Fischer projections:
 D and L Configurations

 d = dextrorotatory
 l = levorotatory
 D, L = relative to glyceraldehyde
 Importance of
Stereochemistry
 All AA’s found in proteins are L geometry
 S enantiomer for all except cysteine

 D-AA’s are found in bacteria

 Geometry of proteins affects reactivity (e.g
binding of substrates in enzymes)
 Thalidomide
 Non-standard Amino Acids

 AA derivatives
 Modification of AA after
protein synthesized
 Terminal residues or R
groups
 Addition of small alkyl
group, hydroxyl, etc.
 D-AAs
 Bacteria
 CHEM 2412 Review

 Carboxylic acid + amine = ?

O O
heat
R C OH + H2N R R C NH R + H2O

 Structure of amino acid

R

H2N C CO2H

H
 The Peptide Bond
 Chain of amino acids = peptide or protein
 Amino acid residues connected by peptide bonds
 Residue = AA – H2O
 The Peptide Bond

 Non-basic and non-acidic in pH 2-12 range

due to delocalization of N lone pair
O O

C
N N

H H
Rigid
restricted rotation

 Amide linkage is planar, NH and CO are anti

 Polypeptides

 Linear polymers (no branches)

 AA monomers linked head to tail
 Terminal residues:
 Free amino group (N-terminus)
 Draw on left
 Free carboxylate group (C-terminus)
 Draw on right
 pKa values of AAs in polypeptides differ
slightly from pKa values of free AAs
 Naming Peptides

 Name from the free amine (NH3+)

 Use -yl endings for the names of the amino acids
 The last amino acid with the free carboxyl group (COO-)
uses its amino acid name

(GA)
 Amino Acid Ambiguity

 Glutamate (Glu/E) vs. Glutamine (Gln/Q)

 Aspartate (Asp/D) vs. Asparagine (Asn/N)
 Converted via hydrolysis
 Use generic abbreviations for either
 Glx/Z
 Asx/B

 X = undetermined or nonstandard AA
 Learning Check

Write the name of the following tetrapeptide using amino

acid names and three-letter abbreviations.
CH3
CH3 S
CH CH3 SH CH2
CH3 O CH O CH2 O CH2 O
-
H3N CH C N CH C N CH C N CH C O
H H H
 Learning Check

 Draw the structural formula of each of the following peptides.

A. Methionylaspartic acid
B. Alanyltryptophan
C. Methionylglutaminyllysine
D. Histidylglycylglutamylalanine
 Outline (part II)

 Sections 3.3 and 3.4

 Separation and purification
 Protein sequencing
 Analysis of primary structure
 Protein size

 In general, proteins contain > 40 residues

 Minimum needed to fold into tertiary structure
 Usually 100-1000 residues
 Percent of each AA varies
 Proteins separated based on differences in
size and composition
 Proteins must be pure to analyze, determine
structure/function
 Factors to control
 pH
 Keep pH stable to avoid denaturation or chemical degradation
 Presence of enzymes
 May affect structure (e.g. proteases/peptidase)
 Temperature
 Control denaturation (0-4°C)
 Control activity of enzymes
 Thiol groups
 Reactive
 Add protecting group to prevent formation of new disulfide bonds
 Exposure to air, water
 Denature or oxidize
 Store under N2 or Ar
 Keep concentration high
 General Separation Procedure

 Detect/quantitate protein (assay)

 Determine a source (tissue)
 Extract protein
 Suspend cell source in buffer
 Homogenize
 Break into fine pieces
 Cells disrupted
 Soluble contents mix with buffer
 Centrifuge to separate soluble and insoluble
 Separate protein of interest
 Based on solubility, size, charge, or binding ability
 Solubility

 Selectively precipitate protein

 Manipulate
 Concentration of salts
 Solvent
 pH
 Temperature
 Concentration of salts

 Adding small amount of salt increases [Protein]

 Salt shields proteins from each other, less
precipitation from aggregation
 Salting-in
 Salting out
 Continue to increase [salt] decreases [protein]
 Different proteins salt out at different [salt]
 Other Solubility Methods

 Solvent
 Similar theory to salting-out
 Add organic solvent (acetone, ethanol) to interact with
water
 Decrease solvating power
 pH
 Proteins are least soluble at pI
 Isoelectric precipitation
 Temperature
 Solubility is temperature dependent
 Chromatography

 Mobile phase
 Mixture dissolved in liquid or
solid
 Stationary phase
 Porous solid matrix
 Components of mixture
pass through the column
at different rates based on
properties
 Types of Chromatography

 Paper
 Stationary phase = filter paper
 Same theory as thin layer chromatography (TLC)
 Components separate based on polarity
 High-performance liquid (HPLC)
 Stationary phase = small uniform particles, large surface area
 Adapt to separate based on polarity, size, etc.
 Hydrophobic Interaction
 Hydrophobic groups on matrix
 Attract hydrophobic portions of protein
 Types of Chromatography

 Ion-exchange
 Stationary phase =
chemically modified to
include charged groups
 Separate based on net
charge of proteins
 Anion exchangers
 Cation groups (protonated
amines) bind anions
 Cation exchangers
 Anion groups (carboxylates)
bind cations
 Types of Chromatography

 Gel-filtration
 Size/molecular exclusion
chromatography
 Stationary phase = gels
with pores of particular
size
 Molecules separate based
on size
 Small molecules caught in
pores
 Large molecules pass
through
 Types of Chromatography

 Affinity
 Matrix chemically
altered to include a
molecule designed
to bind a particular
protein
 Other proteins pass
through
 UV-Vis Spectroscopy

 Absorbance used to
monitor protein
concentrations of each
fraction
  = 280 nm
 Absorbance of aromatic
side groups
 Electrophoresis

 Migration of ions in an electric field

 Electrophoretic mobility (rate of movement) function of
charge, size, voltage, pH
 The positively charged proteins move towards the negative
electrode (cathode)
 The negatively charged proteins move toward the positive
electrode (anode)
 A protein at its pI (neutral) will not migrate in either direction
 Variety of supports (gel, paper, starch, solutions)
 Protein Sequencing

 Determination of primary structure

 Need to know to determine 3D structure
 Gives insight into protein function
 Approach:
 Denature protein
 Break protein into small segments
 Determine sequences of segments

 Animation
 End group analysis

 Identify number of terminal AAs

 Number of chains/subunits
 Identify specific AA

 Dansyl chloride/dabsyl chloride

 Sanger method (FDNB)
 Edman degradation (PITC)

Bovine
insulin
 Dansyl chloride
 Reacts with primary amines
N

 N-terminus O

 Yields dansylated polypeptides + H2N CH C

R
 Dansylated polypeptides SO2

hydrolyzed to liberate the Cl

modified dansyl AA
 Dansyl AA can be identified by
chromatography or
N N
spectroscopy (yellow
fluorescence) HCl +
H3O+
+ other free AAs

 Useful method when protein

SO2 O O
fragmented into shorter HN CH C
SO2

HN CH C OH

polypeptides R R
 Dabsyl chloride and FDNB

 Same result as
O
dansyl chloride
N N

N S Cl

 Dabsyl chloride

 1-Fluoro-2,4-
dinitrobenzene
(FDNB)
 Sanger method
 Edman degradation
 Phenylisothiocyanate (PITC)
 Reacts with N-terminal AA to produce a phenylthiocarbamyl (PTC)
 Treat with TFAA (solvent/catalyst) to cleave N-terminal residue
 Does not hydrolyze other AAs
 Treatment with dilute acid makes more stable organic compound
 Identify using NMR, HPLC, etc.
 Sequenator (entire process for proteins < 100 residues)
 Fragmenting Proteins

 Formation of smaller segments to assist with

sequencing
 Process:
 Cleave protein into specific fragments
 Chemically or enzymatically
 Break disulfide bonds
 Purify fragments
 Sequence fragments
 Determine order of fragments and disulfide bonds
Cleaving Disulfide Bonds

 Oxidize with performic acid

O

H C O OH

 Cys residues form cysteic acid

 Acid can oxidize other
residues, so not ideal
 Cleaving Disulfide Bonds

 Reduce by mercaptans (-SH)

 2-Mercaptoethanol
 HSCH2CH2OH
 Dithiothreitol (DTT)
 HSCH2CH(OH)CH(OH)CH2SH
 Reform cysteine residues
 Oxidize thiol groups with
iodoacetete (ICH2CO2-) to
prevent reformation of disulfide
bonds
 Hydrolysis

 Cleaves all peptide bonds

 Achieved by
 Enzyme
 Acid
 Base
 After cleavage:
 Identify using chromatography
 Quantify using absorbance or fluorescence
 Disadvantages
 Doesn’t give exact sequence, only AAs present
 Acid and base can degrade/modify other residues
 Enzymes (which are proteins) can also cleave and affect results
 Enzymatic and Chemical Cleavage

 Enzymatic
 Enzymes used to break
protein into smaller peptides
 Endopeptidases
 Catalyze hydrolysis of
internal peptide bonds

 Chemical
 Chemical reagents used to
break up polypeptides
 Cyanogen bromide (BrCN)
An example
 Another example
 A protein is cleaved with cyanogen bromide to yield the
following sequences:
 Arg-Ala-Tyr-Gly-Asn
 Leu-Phe-Met
 Asp-Met
 The same protein is cleaved with chymotrypsin to yield the
following sequences:
 Met-Arg-Ala-Tyr
 Asp-Met-Leu-Phe
 Gly-Asn
 What is the sequence of the protein?
 Suggested Problems, Chapter 3

 1-5, 7, 10-13, 15, 18