Chapter 3: Amino Acids, Peptides, and Proteins

Dr. Rajabi

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

The building blocks of proteins Also used as single molecules in biochemical

pathways 20 standard amino acids (-amino acids)( 21

amino acid=Selenocysteine) 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

A good buffer at ~ pH 6.

pI =

Histidine

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

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 structure:There are four levels of protein structure (primary, secondary, tertiary and quaternary) Primary structure: •  The primary structure of a protein is its unique sequence of amino acids.

– Lysozyme, an enzyme that attacks bacteria, consists of a polypeptide chain of 129 amino acids.

– The precise primary structure of a protein is determined by inherited genetic information.

– At one end is an amino acid with a free amino group the (the N-terminus) and at the other is an amino acid with a free carboxyl group the (the C-terminus).

2- Secondary structure: Results from hydrogen bond

formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen of another peptide bond. According to H-bonding there are two main forms of secondary structure:

α-helix: It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one

β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment).

I →I+4

Hydrogen bonding in α-helix: In the α-helix CO of the one amino acid residue forms H-bond with NH of the forth one.

Supersecondary structure or Motifs : occurs by combining secondary structure.

The combination may be: α-helix- turn- α-helix- turn…..etc Or: β-sheet -turn- β-sheet-turn………etc

Or: α-helix- turn- β-sheet-turn- α-helixTurn (or bend): is short segment of polypeptides (3-4 amino acids)

that connects successive secondary structures.e.g. β-turn: is small polypeptide that connects successive strands of β-

sheets.

b. Strong covalent bonds include disulfide bridges, that form between the sulfhydryl groups (SH) of cysteine monomers, stabilize the structure.

• Quaternary structure: results from the aggregation (combination) of two or more polypeptide subunits held together by non-covalent interaction like H-bonds, ionic or hydrophobic interactions.

• Examples on protein having quaternary structure:

– Collagen is a fibrous protein of three polypeptides (trimeric) that are supercoiled like a rope.

•This provides the structural strength for their role in connective tissue.

– Hemoglobin is a globular protein with four polypeptide chains (tetrameric)

– Insulin : two polypeptide chains (dimeric)

Summary of Protein Structures

Copyright © 2007 by Pearson Education, Inc Publishing as Benjamin Cummings

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

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

2- Secondary structure: Results from hydrogen bond

formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen of another peptide bond. According to H-bonding there are two main forms of secondary structure:

α-helix: It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one

β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment).