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 (a-amino acids)

Two functional groups:



carboxylic acid group
amino group on the alpha (a) carbon
Have different side groups (R)

Properties dictate behavior of AAs
R side chain
|
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



R group = carboxylic
acid
Donates H+
Negatively charged

Basic




R group = amine
Accepts H+
Positively 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
organization chart
pH and Ionization

Consider glycine:
O
H3N
CH
C
O
OH
O
OH-
OH-
H3N
CH
C
O
+
H3O
H3O
H
Glycine ion at
acidic pH
(charge = 1+)

H2N
H
Zwitterion of glycine
(charge = 0)
CH
C
+
H
Glycine ion at
basic pH
(charge = 1-)
Note that the uncharged species never forms
O
Titration of Glycine

pK1


pK2



[cation] = [zwitterion]
[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
pK1
H3N
CH
C
OH
CH2CH2CH2CH2NH3+
O
pK2
H3N
CH
C
O
pKR
H2N
CH2CH2CH2CH2NH3+
CH
C
O
CH2CH2CH2CH2NH3+

Which species is the isoelectric form?

So, pI = ½ (pK2 + pKR)
H2N
CH
C
CH2CH2CH2CH2NH2
= ½ (8.95 + 10.53) = 9.74

O
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
H3N
CH
C
O
O
H3N
CH
C
O
OH
H2N
CH
CH2
CH2
CH2
OH
OH
OH
C
O
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
Structure of amino acid
R
H2N
C
H
CO2H
C
NH
R
+
H2O
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
H

N
Rigid
restricted rotation
H
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

Free carboxylate group (C-terminus)
 Draw

on left
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
H3N
S
CH CH3
SH
CH2
CH3 O
CH O
CH2 O
CH2 O
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


Presence of enzymes



Control denaturation (0-4°C)
Control activity of enzymes
Thiol groups



May affect structure (e.g. proteases/peptidase)
Temperature


Keep pH stable to avoid denaturation or chemical degradation
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 out


Salting-in
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

l = 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

Yields dansylated polypeptides

Dansylated polypeptides
hydrolyzed to liberate the
modified dansyl AA


Dansyl AA can be identified by
chromatography or
spectroscopy (yellow
fluorescence)
Useful method when protein
fragmented into shorter
polypeptides
O
+
H2N
CH
C
R
SO2
Cl
N
N
H3O+
HCl
+
+ other free AAs
O
SO2
HN
CH
R
C
O
SO2
HN
CH
R
C
OH
Dabsyl chloride and FDNB

Same result as
dansyl chloride
N
O
N
N
S
O

Dabsyl chloride

1-Fluoro-2,4dinitrobenzene
(FDNB)

Sanger method
Cl
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




After cleavage:



Enzyme
Acid
Base
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
Fundamentals of Protein
Structure
Our life is maintained by
molecular network systems
Molecular network
system in a cell
(From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)
Proteins play key roles in a
living system

Three examples of protein functions

Alcohol
dehydrogenase
oxidizes alcohols
to aldehydes or
ketones
Catalysis:
Almost all chemical reactions in a
living cell are catalyzed by protein
enzymes.

Transport:
Some proteins transports various
substances, such as oxygen, ions,
and so on.

Information transfer:
For example, hormones.
Haemoglobin
carries oxygen
Insulin controls
the amount of
sugar in the
blood
Amino acid: Basic unit of
protein
R
NH3
+
C
Amino group
H
Different side chains,
R, determin the
COO
properties of 20
Carboxylic
amino acids.
acid group
An amino acid
20 Amino acids
Glycine (G)
Alanine (A)
Valine (V)
Isoleucine (I)
Leucine (L)
Proline (P)
Methionine (M)
Phenylalanine (F)
Tryptophan (W)
Asparagine (N)
Glutamine (Q)
Serine (S)
Threonine (T)
Tyrosine (Y)
Cysteine (C)
Lysine (K)
Arginine (R)
Histidine (H)
Asparatic acid (D) Glutamic acid (E)
White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic
Proteins are linear polymers of
amino acids
R1
R2
NH3＋ C COO ＋ NH3＋ C COO ＋
ー
ー
H
H
H 2O
A carboxylic acid
condenses with an amino
group with the release of a
water
H 2O
R1
R2
R3
NH3＋ C CO NH C CO NH C CO
H
A
F
Peptide
bond
G
N
S
Peptide
bond
H
T
D
K
G
H
S
A
The amino acid
sequence is called as
primary structure
Amino acid sequence is encoded
by DNA base sequence in a gene
DNA
molecule
＝
・
G
C
G
C
T
T
A
A
G
C
G
C
・
・ DNA base
C
G sequence
C
G
A
A
T
T
C
G
C
G
・
Amino acid sequence is encoded
by DNA base sequence in a gene
T
T
A
G
Phe
Leu
Leu
Ile
Met
Val
TCT
TCC
TCA
TCG
CCT
CCC
CCA
CCG
ACT
ACC
ACA
ACG
GCT
GCC
GCA
GCG
Ser
Pro
Thr
Ala
TAT
TAC
TAA
TAG
CAT
CAC
CAA
CAG
AAT
AAC
AAA
AAG
GAT
GAC
GAA
GAG
G
Tyr
Stop
His
Gln
Asn
Lys
Asp
Glu
TGT
TGC
TGA
TGG
CGT
CGC
CGA
CGG
AGT
AGC
AGA
AGG
GGT
GGC
GGA
GGG
Cys
Stop
Trp
Arg
Ser
Arg
Gly
T
C
A
G
T
C
A
G
T
C
A
G
T
C
A
G
Third letter
First letter
C
TTT
TTC
TTA
TTG
CTT
CTC
CTA
CTG
ATT
ATC
ATA
ATG
GTT
GTC
GTA
GTG
C
Second letter
A
Gene is protein’s blueprint,
genome is life’s blueprint
DNA
Genome
Gene
Protein
Gene Gene Gene Gene
Gene
Gene
Gene
Gene
Gene
Gene
Gene Gene Gene
Gene
Protein
Protein
Protein
Protein
Protein Protein
Protein
Protein
Protein
Protein Protein
Protein
Protein
Protein
Gene is protein’s blueprint,
genome is life’s blueprint
Glycolysis network
Genome
Gene Gene Gene Gene
Gene
Gene
Gene
Gene
Gene
Gene
Gene Gene Gene
Gene
Protein
Protein
Protein
Protein
Protein Protein
Protein
Protein
Protein
Protein Protein
Protein
Protein
Protein





In 2003, Human genome
sequence was deciphered!
Genome is the complete set of genes of a living thing.
In 2003, the human genome sequencing was completed.
The human genome contains about 3 billion base pairs.
The number of genes is estimated to be between 20,000 to
25,000.
The difference between the genome of human and that of
chimpanzee is only 1.23%!
3 billion base pair => 6 G letters
&
1 letter => 1 byte
The whole genome can be recorded in
just 10 CD-ROMs!
Each Protein has a unique
structure
Amino acid sequence
NLKTEWPELVGKSVEE
AKKVILQDKPEAQIIVL
PVGTIVTMEYRIDRVR
LFVDKLDNIAEVPRVG
Folding!
Basic structural units of proteins:
Secondary structure
α-helix
β-sheet
Secondary structures, α-helix
and β-sheet, have regular
hydrogen-bonding patterns.
Three-dimensional structure of
proteins
Tertiary
structure
Quaternary structure
Hierarchical nature of protein
structure
Primary structure (Amino acid sequence)
↓
Secondary structure （α-helix, β-sheet）
↓
Tertiary structure （Three-dimensional structure
formed by assembly of secondary structures）
↓
Quaternary structure （Structure formed by more
than one polypeptide chains）
Close relationship between
protein structure Hormone
and its
function
Antibody
Example of enzyme reaction
receptor
substrates
A
enzyme
enzyme
B
Matching
the shape
to A
enzyme
A
Binding to A
Digestion
of A!
Protein structure prediction has
remained elusive over half a
century
“Can we predict a protein structure from
its amino acid sequence?”
Now, impossible!






Summary
Proteins are key players in our living systems.
Proteins are polymers consisting of 20 kinds of amino
acids.
Each protein folds into a unique three-dimensional structure
defined by its amino acid sequence.
Protein structure has a hierarchical nature.
Protein structure is closely related to its function.
Protein structure prediction is a grand challenge of
computational biology.