Amino Acids, Peptides, and Proteins

Outline

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)

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

Nonpolar Amino Acids

Hydrophobic, neutral, aliphatic

Polar Amino Acids

Hydrophilic, neutral, typically H-bond

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

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

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 pKas)

AAs also have multiple pKas 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

pH and Ionization

Consider glycine:
O

H3N

CH

Glycine ion at
acidic pH
(charge = 1+)

O
OH

OHH3O

H3N

CH

O
O

OHH3O

Zwitterion of glycine
(charge = 0)

H2N

CH

Glycine ion at
basic pH
(charge = 1-)

Note that the uncharged species never forms

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 pKas = (pK1 + pK2)

pIglycine = (2.34 + 9.60) = 5.97

Animation

pI of Lysine

For AAs with 3 pKas, pI = average of two relevant pKa values

Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53):

O

H3N

CH

O
OH

CH2CH2CH2CH2NH3+

pK1

pK2
H3N

CH

pKR
H2N

CH2CH2CH2CH2NH3+

CH

CH2CH2CH2CH2NH3+

Which species is the isoelectric form?

So, pI = (pK2 + pKR)

H2N

CH

CH2CH2CH2CH2NH2

= (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
H3N

CH

O
O

H3N

CH

O
OH

H2N

CH

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 AAs found in proteins are L geometry

S enantiomer for all except cysteine

D-AAs 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

Organic Chem Review

Carboxylic acid + amine = ?

O

O
heat

OH

H2N

Structure of amino acid

R
H2N

C
H

CO2H

NH

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

C
N
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

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

CH CH3

SH

CH2

CH3 O

CH O

CH2 O

CH2 O

CH C N

CH C N

CH C N

CH C O

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 (0-4C)

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

= 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

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-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

H2N

CH

R
SO2
Cl

HCl

H3O+

+ other free AAs

SO2
HN

CH
R

SO2
HN

CH
R

OH

Dabsyl chloride and FDNB

Same result as
dansyl chloride

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

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

Doesnt 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

PRIMARY STRUCTURE
The sequence of amino acids
MIL1 sequence:
>gi|7662506|ref|NP_056182.1| MIL1 protein [Homo sapiens]
MEDCLAHLGEKVSQELKEPLHKALQMLLSQPVTYQAFRECTLETTVHASGWNKILVPLVLLRQMLL
ELTRLGQEPLSALLQFGVTYLEDYSAEYIIQQGGWGTVFSLESEEEEYPGITAEDSNDIYILPSDN
SGQVSPPESPTVTTSWQSESLPVSLSASQSWHTESLPVSLGPESWQQIAMDPEEVKSLDSNGAGEK
SENNSSNSDIVHVEKEEVPEGMEEAAVASVVLPARELQEALPEAPAPLLPHITATSLLGTREPDTE
VITVEKSSPATSLFVELDEEEVKAATTEPTEVEEVVPALEPTETLLSEKEINAREESLVEELSPAS
EKKPVPPSEGKSRLSPAGEMKPMPLSEGKSILLFGGAAAVAILAVAIGVALALRKK
length: 386amino acids

Anne-Marie Ternes

PRIMARY STRUCTURE

The numbers of amino acids vary

(e.g. insulin 51, lysozyme 129, hemoglobin 574,
gamma globulin 1250)
The primary structure determines the folding of
the polypeptide to give a functional protein
Polar amino acids (acidic, basic and neutral)
are hydrophilic and tend to be placed on the
outside of the protein.
Non-polar (hydrophobic) amino acids tend to be
placed on the inside of the protein

2007 Paul Billiet ODWS

SECONDARY STRUCTURE
The folding of the N-CC backbone of the
polypeptide chain
using weak hydrogen
bonds

Text 2007 Paul Billiet ODWS

Science Student

SECONDARY STRUCTURE

This produces the alpha helix and beta pleating

The length of the helix or pleat is determined by certain amino acids that
will not participate in these structures
(e.g. proline)

Text2007 Paul Billiet ODWS

Dr Gary Kaiser

TERTIARY STRUCTURE
The folding of the polypeptide into
domains whose chemical properties are
determined by the amino acids in the
chain

MIL1 protein

2007 Paul Billiet ODWS

Anne-Marie Ternes

TERTIARY STRUCTURE

This folding is sometimes held together by strong

covalent bonds
(e.g. cysteine-cysteine disulphide bridge)
Bending of the chain takes place at certain amino
acids
(e.g. proline)
Hydrophobic amino acids tend to arrange
themselves inside the molecule
Hydrophilic amino acids arrange themselves on
the outside

2007 Paul Billiet ODWS

Chain B of Protein Kinase C

Max Planck Institute for Molecular Genetics

When the polypeptide folds into a threedimensional shape, it is called a protein

The three-dimensional shape of a protein is

called
its tertiary structure
Myoglobin
Binds oxygen
Found in the muscles
Acts as a storage site
for oxygen
Makes up the dark meat in
chicken

QUATERNARY STRUCTURE
Some proteins are
made of several
polypeptide subunits
(e.g. hemoglobin has
four)

Protein Kinase C
Max Planck Institute for Molecular Genetics
Text 2007 Paul Billiet ODWS

QUATERNARY STRUCTURE
These subunits fit together to form the
functional protein
Therefore, the sequence of the amino acids
in the primary structure will influence the
protein's structure at two, three or more
levels

2007 Paul Billiet ODWS

Shape of the protein is important for its

function
Ex. Insulin = 51 amino
acids

Shape of the protein is important for its

function
Ex. Insulin = 51 amino
acids

Result
Protein structure depends upon the amino
acid sequence
This, in turn, depends upon the sequence
of bases in the gene

2007 Paul Billiet ODWS

PROTEIN FUNCTIONS
Protein structure determines protein function
Denaturation or inhibition which may change
protein structure will change its function
Coenzymes and cofactors in general may
enhance the protein's structure

2007 Paul Billiet ODWS

Types of Proteins
Type

Function

Communication

Cell signaling
Ex. Hormones in the bloodstream

Defense

Protection from infection

Ex. Antibodies in the bloodstream

Structure

Mechanical support
Ex. Collagen in skin & keratin in hair/nails

Storage

Stores nutrients
Ex. Albumin in egg whites

Contractile

Movement
Ex. Actin and myosin in muscles

Transport

Carries other molecules

Ex. Hemoglobin

Hormones

Chemical messengers
Ex. Growth hormone stimulates bone growth

Enzymes

Speed up chemical reactions

Ex. Catalase

Fibrous proteins
Involved in structure: tendons ligaments
blood clots
(e.g. collagen and keratin)
Contractile proteins in movement: muscle,
microtubules
(cytoskelton, mitotic spindle, cilia, flagella)

2007 Paul Billiet ODWS

Just for fun facts:

Your hair is composed of all -helix
Spider webs are all -pleated sheets

Globular proteins
most proteins which move around (e.g.
albumin, casein in milk)
Proteins with binding sites:
enzymes, hemoglobin, immunoglobulins,
membrane receptor sites

2007 Paul Billiet ODWS

Antibodies are Produced by B

Lymphocytes

Antibodies are Proteins that Recognize Specific

Antigens

Epitopes: Antigen Regions that

Interact with Antibodies