Amino Acids, Peptides, and Proteins

Outline

Amino Acids

Chemical structure

Acid-base properties

Stereochemistry

Non-standard amino acids

Formation of Peptide Bonds

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

Amino Acids

R side chain

| H2N— C —COOH

|H

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

Zwitterions

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

Remember H3PO4 (multiple pKa’s)

AAs also have multiple pKa’s due to multiple ionizable

groups

Acid-base Properties

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:

Note that the uncharged species never forms

H3N CH C

H

OH

O

H3N CH C

H

O

O

H2N CH C

H

O

O

OH-

H3O+

OH-

H3O+

Glycine ion at acidic pH

(charge = 1+)

Zwitterion of glycine (charge = 0)

Glycine ion at basic pH

(charge = 1-)

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):

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)

H3N CH C

CH2CH2CH2CH2NH3+

OH

O

H3N CH C

CH2CH2CH2CH2NH3+

O

O

H2N CH C

CH2CH2CH2CH2NH3+

O

O

H2N CH C

CH2CH2CH2CH2NH2

O

O

pK1 pK2 pKR

Learning Check

Would the following ions of serine exist at a pH above, below, or at pI?

H3N CH C

CH2

O

O

OH

H3N CH C

CH2

OH

O

OH

H2N CH C

CH2

O

O

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 = ?

Structure of amino acid

R C OH

O

+ H2N R R C NH

O

+ H2Oheat

R

H2N C CO2H

H

R

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

Amide linkage is planar, NH and CO are anti

C

O

N

H

O

N

HRigid

restricted rotation

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

Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.

Learning Check

CH CH3

CH3

H3N CH C

O

N

H

CH C

O

N

H

CH C

O

N

H

CH C O-

OCH CH2

CH2

S

CH3

CH2

SH

CH3

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-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

N

SO2

Cl

+

HN CH

R

C

O

N

SO2

H2N CH

R

C

O

HCl +H3O+

HN CH

R

C

O

OH

N

SO2

+ other free AAs

Dabsyl chloride and FDNB

Same result as dansyl chloride

Dabsyl chloride

1-Fluoro-2,4-dinitrobenzene (FDNB) Sanger method

SN

NN O

O

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

Cys residues form cysteic acid

Acid can oxidize other

residues, so not ideal

H C

O

O OH

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

PRIMARY STRUCTURE

The sequence of amino acids

MIL1 sequence:>gi|7662506|ref|NP_056182.1| MIL1 protein [Homo sapiens]MEDCLAHLGEKVSQELKEPLHKALQMLLSQPVTYQAFRECTLETTVHASGWNKILVPLVLLRQMLLELTRLGQEPLSALLQFGVTYLEDYSAEYIIQQGGWGTVFSLESEEEEYPGITAEDSNDIYILPSDNSGQVSPPESPTVTTSWQSESLPVSLSASQSWHTESLPVSLGPESWQQIAMDPEEVKSLDSNGAGEKSENNSSNSDIVHVEKEEVPEGMEEAAVASVVLPARELQEALPEAPAPLLPHITATSLLGTREPDTEVITVEKSSPATSLFVELDEEEVKAATTEPTEVEEVVPALEPTETLLSEKEINAREESLVEELSPASEKKPVPPSEGKSRLSPAGEMKPMPLSEGKSILLFGGAAAVAILAVAIGVALALRKK

length: 386amino acids © Anne-Marie Ternes

PRIMARY STRUCTURE The numbers of amino acids vary

(e.g. insulin 51, lysozyme 129, haemoglobin 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

Infinite variety

The number of possible sequences is infinite An average protein has 300 amino acids, At each position there could be one of 20 different amino acids = 10390 possible combinations

Most are uselessNatural selection picks out the best

© 2007 Paul Billiet ODWS

SECONDARY STRUCTURE

The folding of the N-C-C backbone of the polypeptide chain using weak hydrogen bonds

© Science Student

© Text 2007 Paul Billiet ODWS

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)

© Dr Gary Kaiser © Text2007 Paul Billiet ODWS

TERTIARY STRUCTURE

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

MIL1 protein

© Anne-Marie Ternes © 2007 Paul Billiet ODWS

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

© Max Planck Institute for Molecular GeneticsChain B of Protein Kinase C

When the polypeptide folds into a three-

dimensional 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. haemoglobin 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 ProteinsTypeType FunctionFunctionCommunication Cell signaling

Ex. Hormones in the bloodstream

Defense Protection from infectionEx. Antibodies in the bloodstream

Structure Mechanical supportEx. Collagen in skin & keratin in hair/nails

Storage Stores nutrientsEx. Albumin in egg whites

Contractile MovementEx. Actin and myosin in muscles

Transport Carries other moleculesEx. Hemoglobin

Hormones Chemical messengersEx. Growth hormone stimulates bone growth

Enzymes Speed up chemical reactionsEx. 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. albumen, casein in milk)

Proteins with binding sites: enzymes, haemoglobin, 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