Proteins

Amino acids-proteins

• I. Overview• Most diverse and abundant molecules in living

systems• Functional components: enzymes, hormones, cell-

surface receptors• Structural components: cell membranes, organelles;

bone, skin, muscle, connective tissue• Other specialized roles: immunoglobulins,

hemoglobin, albumin

II. Structure of Amino Acids

• More than 300 amino acids known, but only 20 coded for by DNA

• At pH 7.4 (physiological pH), amino acids exist in zwitterionic form (positive NH3+ and negative COO- charges).

• Classified based on side chain (R) group: Nonpolar, Polar, Charged (acidic or basic)

A. Amino acids with non-polar side chains

• do not bind nor give protons• do not form hydrogen bonds• have hydrophobic interactions

• 1. Location of non-polar (hydrophobic) amino acids in proteins– In soluble proteins (aqueous environment), found in

the interior of proteins (shielded from environment)– In membranes or other hydrophobic environments,

found on protein surface. – Proline: side chain forms an imino group

B. Amino acids with uncharged polar side chains

• 0 charge at neutral pH• Cys & Tyr can lose a proton at alkaline pH• Ser, Thr & Tyr – polar –OH can form hydrogen

bonds • Asn & Gln contain –COOH (carboxy) and –

CONH2 (carboxyamine) groups – can form hydrogen bonds.

• 1. Disulfide bond:• Side chain of Cys contains –SH group –

important active site of enzymes• Proteins with 2 –SH groups can form a

disulphide bridge or cystine dimer (-S-S- , intermolecular or intramolecular).

B. Amino acids with uncharged polar side chains

• 2. Side chains as sites of attachments for other compounds:

• Ser, Thr & Tyr contain polar –OH group – site of attachment for PO4- group, for e.g. Ser side-chain important active site component in many enzymes– -CONH2 group of Asn and –OH group of Ser &

Thr serve as site of attachment of oligosaccharide chains in glycoproteins

C. Amino acids with acidic side chains

• Asp & Glu are proton donors.• At neutral pH (physiological), side chains fully

ionized or dissociated (COO-) and carry a net negative charge.

• Contribute a negative charge to proteins .• Aspartate (aspartic acid) and glutamate

(glutamic acid).• R groups typically have a pK< 7

D. Amino acids with basic side chains

• Side chains of basic amino acids accept protons • At physiologic pH, side chains of Lys and Arg are fully ionized –

positively charged ( NH3+)• Contribute a positive charge to proteins that contain them• Have a pK value>7( histones have an abundance of Arg and lys, net +ve charge)

• His -- weakly basic and partially positively charged at physiologic pH- good buffering capacity

• In proteins, can be +ve or –ve depending on environment of protein (important role in proteins like myoglobin).

E. Abbreviations and symbols for commonly occurring amino acids3-letter abbreviation and one-letter symbol1. Unique first letter

CysteineCys C

Histidine His H

Isoleucine Ile I

Methionine Met M

Serine Ser S

Valine Val V

AlanineAla A

Glycine Gly G

Leucine Leu L

Proline Pro P

Threonine Thr T

2. Most commonly occurring amino acids have priority

3. Similar sounding names

ArginineArg R (“aRginine)

Asparagine Asn N (contains N)

Aspartate Asp D (“asparDic”)

Glutamate Glu E (“glutEmate”)

Glutamine Gln Q (“Q-tamine”)

Phenylalanine

Phe F (“Fenylalanine”)

Tyrosine Tyr Y (“tYrosine”)

Tryptophan Trp W (double ring in the molecule)

Aspartate or asparagines

Asx B

Glutamate or glutamine

Glx Z

Lysine Lys K (near L)

Undetermined amino acid

X

4. Letter close to initial letter:

F. Optical properties of amino acids:

• α-C of each amino acid attached to 4 different chemical groups

• α-C is chiral or optically active i.e. it has four different groups attached to the -carbon (except Gly). The number of optical isomers is 2n, where n is the number of chiral atoms in the molecule.

• 2 stereoisomers, optical isomers or enantiomers: D- and L- forms are mirror images of one another, only L-forms found in human bodies

I. Overview

• 20 amino acids linked together with peptide bonds

• 4 organizational levels: primary, secondary, tertiary and quaternary

Primary Structure

• Primary Structure of Proteins • Sequence of amino acids = primary

structure• Genetic diseases result from proteins with

abnormal sequences

Primary structure: insulin

Peptide Bond

• Not broken when proteins are denatured

• Prolonged exposure to acid or base at high temps is necessary to break bonds.

• 1. Naming the peptide• a. order of amino acids in a peptide• Left (N-terminal a.a.) is written first, C-terminal next• b. Naming of polypeptides• component a.a. in peptides called moieties or

residues.• Except C-terminal, all moieties called –yl instead of –

ine –ate, or -ic• E.g. valylglycylleucine

Characteristics of the peptide bond:

• a. Lack of rotation around the bond:• partial double bond- rigid and planar. bond

between -C and -amino or –CO group is rotatable

• b. Trans configuration:• (steric interference in cis position) • c. Uncharged but polar:• like all –CONH2 links, peptide bonds do not

protonate between pH 2-12• only side chains and N- and C- terminals can ionize• peptide bond is polar (uncharged) and can be

involved in H-bonding.

Characteristics of the peptide bond

3. Trans configuration

• minimizes steric hindrance

A peptide bond is formed from a condensation reaction (dehydration) involving two amino acids.A molecule of H2O is eliminated.

H3N C C

H

CH3

alanine

O

O

valine

N C C

H

CH

H3C CH3

O

O

H

H

H

H3N C C

H

CH3

alaninylvaline

N C C

H

CH

H3C CH3

O O

OH

H2O

Dipeptide formation

peptide bond

amino terminus(a amino group)

carboxyl terminusa a

bb

g g

H3N C C

H

R1

N C C

H

R2

O O

OH

Characteristics of the peptide bond

Characteristics of the peptide bond

1. partial double-bond character

• due to resonance

H3NC

CH

R1

NC

C

R2

H

O

O

OHH3N

CC

H

R1

NC

C

R2

H

O

O

OH

H3N C C

H

R1

N C C

H

R2

O O

OH

H3NC

CH

R1

NC

C

R2

H

O

O

OH

Characteristics of the peptide bond

2. rigid and planar

• rotation occurs around single bonds but not around double bonds

H3N C C

H

R1

N C C

H

R2

OO

OH

no rotation around peptide bond

H3NC

CH

R1

NC

CH

R2

OO

O

H

H3NC

CH

R1

NC

C

R2

H

O

O

OH

HC

CR1

NH3

NC

C

R2

H

O

O

OH

Rotation around single bonds

Because no rotation is possible around double bonds, the stereochemistry of the peptide bond is fixed.

H3NC

CH

R1

NC

C

R2

H

O

O

OH

4. Uncharged but polar

• dipole moment exists due to separation of charge

Characteristics of the peptide bond

Characteristics of the peptide bond - summary• partial double bond character

• trans configuration

• uncharged but polar

• rigid and planar

H3NC

CH

R1

NC

C

R2

H

O

O

OH

amide planetrans

config

B. Determination of the amino acid composition of a polypeptide

• First, identify and quantify constituent amino acids.• Pure sample must be used, contamination gives

errors. • 1. Acid hydrolysis:• Hydrolyzed by strong acid at 110 C for 24 h• Peptide bonds cleaved• Gln & Asn Glu & Asp; Trp mostly destroyed• Procedure gives composition but not sequence

• 2. Chromatography:• Individual aa’s separated by cation-exchange chromatography • Anion-exchange resin for -vely charged aa’s• Eluted from column by buffers of increasing ionic strength and pH• aa’s separated at different ionic strength and pH• 3. Quantitative analysis:• Quantified with ninhydrin purple compd. with amino acids, NH3 and

amines (yellow color with imino group of Pro).• Intensity of color measured in spectrophotometer• Area under curve proportional to amount of amino acid• If MW of protein known, no. of residues of each aa known, otherwise,

only ratio of no. of molecules of each amino acid determined. • Done using amino acid analyzer

C. Sequencing of the peptide from its N-terminal end

• Phenylisothiocyanate – Edman’s reagent – used to label N-terminal res under mildly alkaline conditions. phenylthiohydantoin (PTH).

• This makes N-terminal residue peptide bond weak; break it without breaking others.

• Above process occurs in a cycle to sequence peptide using “sequenator”

• Can be used for polypeptides of 100 a.a. or less.

H2N CH

CH3

C

O

Lys His Phe Leu Arg COOH

1. LabelingN C S

Phenylisothiocyanate (Edman’s reagent)

N-terminal alanine

HN CH

CH3

C

O

Lys His Phe Leu Arg COOH

N

CS

H

labeled peptide

H

2. Acid hydrolysis

cyclization and expulsion of shortened peptide chain

His Phe Leu Arg COOHH2N CH

(CH2)4

C

O

NH2

N-terminal lysine

NC

NHCHC

S

CH3O

+

PTH-alanine

Cleavage of peptide into smaller fragments

• necessary if peptide is > 100 amino acids in length

• occurs before Edman degradation

• need to use more than one cleaving agent in order to determine amino acid sequence • different enzyme/chemical specificity

• overlap peptide fragments in order to determine original sequence

• 2. Chemical Cleavage:• Cyanogen bromide cleaves polypeptides on –CO side of methionine

residue

• 3. Overlapping peptides:• Individual peptides sequenced by Edman’s degradation• Overlapping peptides help determine sequence

• 4. Multimeric proteins:• Multiple peptides separated (H-bonds and noncovalent bonds) by urea or

guanidine.HCl• Disulfide bridges broken with performic acid.

Secondary Structure

Secondary structures include:

• Helical Structures

• Beta Structure (maximally extended primary sequence)

• Random chain (nonrepetitive)

• Secondary structures result from local arrangement of adjacent amino acids into an organized 3- dimensional structure.

• H-bonds are key to stabilizing these structures.

Right-hand a helix

Left-hand a helix

a Helix

Intrachain Hydrogen Bonding is important in maintaining secondary protein structure. Here (in the α helix) the carbonyl oxygen from one amino acid is H-bonded to an alpha nitrogen of the 4th distant amino acid in the polymer.

Hydrogen bond

• 3.6 residues per turn

• R groups extend outward

a helix is disrupted by:

1) P and G

2) large numbers of charged aa’s

3) aa’s with bulky R groups

Myoglobin

b Sheet

• “pleated”

• all peptide bond components involved in H-bonding

• strands visualized as broad arrows

• may be parallel or antiparallel

C terminal

N terminal

b Sheet

b-Bend

• function to reverse the direction of polypeptide chain

• often include charged residues

• usually composed of 4 amino acids including Pro and Gly

• stabilized by ionic and/or H-bonds

Supersecondary structure (motif)• result from local folding of secondary structures into small, discrete, commonly-observed aggregates of secondary structures:

• - - b a b loop

• -a a corner

• extended super secondary structures are known as domains

• b barrel

• twisted b sheet

Tertiary Structure

• Tertiary structure is the 3 dimensional form of a molecule resulting from distant protein-protein interactions within the same polypeptide chain (caused by folding of secondary structures):

Globular proteins are characterized as generally having:

• a variety of different kinds of secondary structure• spherical shape

• good water solubility

• a catalytic/regulatory/transport role i.e. a dynamic metabolic function

• IV. Tertiary Structure of Globular Proteins• Tertiary structure – folding of domains and final

arrangement of domains in protein• Compact, hydrophobic side chains buried in interior• Maximum hydrogen bonding of hydrophilic groups

within molecule

Fibrous proteins are characterized as generally having:

• one dominating kind of secondary structure (i.e. collagen helix in collagen)

• a long narrow rod-like structure

• low water solubility

• a role in determining tissue/cellular structure and function (e.g. collagen, -a keratin)

Interactions involved in maintaining tertiary structure

Hydrogen bonds

Disulfide bonds

Hydrophobic interactions (Van der Waals forces)

Electrostatic interactions (ionic and/or polar interactions)

2 cysteine (-SH) cystine (-S-S)-

-N-H• • •N--N-H• • •O--O-H• • •O--O-H• • •N-

• Domains• Fundamental functional and 3-D structural units

of polypeptides• >200 amino acids 2 or more domains• folding within domain independent of folding

within other domains• each domain has characteristics of small,

compact, globular protein

• 1. Disulfide bonds:• formed from –SH groups of two

cysteine residues Cystine• two Cys may be close by or far

away• stabilize the protein found in

many secreted proteins• 2. Hydrophobic interactions:• interactions between nonpolar

side chains of amino acids in interior of protein

• 3. Hydrogen bonds:• interactions between

polar side chains • interactions between

polar side chains and water enhanced solubility

• 4. Ionic interactions:• e.g. Interaction of –COO-

of Asp with NH3+ of Lys

Protein folding

• Trial and error process that depends on• Composition of side chains• H-bonding• Disulfide bonds• Ionic interactions• To result in most stable or favorable structure• Chaperones: play a role in folding of proteins during

their synthesis (separate, enhance the rate, protect residues).

Denaturation of Proteins

• Destruction of all but primary structure• Denaturing agents: heat, organic solvents,

mechanical shearing, heavy metals, detergents, chaotropic agents

• May be reversible or irreversible• Loss of biological activity

Most proteins do not revert to their original tertiary structures after denaturation.

Ribonuclease enzyme is an exception.

Protein misfolding

• Spontaneous• Mutation• Proteolytic cleavage, e.g. accumulation of amyloid

plaques (amyloid-β) in Alzheimer’s. • Abnomal form of tau accumulation in neufibillary

tangles of Alzheimer’s brain• Prion disease: Creutzfeldt-Jakob disease – humans• A protein- as a degenerative agent

b-Sheet in fibrous (Amyloid) protein

• Amyloid protein deposited in brains of Alzheimer’s disease patients – twisted -pleated sheet fibrils with 3-D structure virtually identical to silk fibrils

Prion

Quaternary Structure

Quaternary structure consists of the association of multimeric proteins (identical or nonidentical) held together by one or more of the following noncovalent interactions:

Hydrogen bonds

Hydrophobic interactions (Van der Waals forces)

Electrostatic interactions (ionic and/or polar)

Hemoglobin