Amino Acids and Proteins

Muhammad Jawad HassanAssistant Professor

Biochemistry

Objectives

• Structure and Classification of amino acids• Peptide Bond and Primary structure of protein • Secondary Structure of protein, Helices and

Sheets• Tertiary and Quaternary Structure of protein,

domain and motifs• Structure-function relationship of proteins and

disease

3

Structure dictates function

Protein structureallows DNAreplication withoutdissociation ofreplicatingmachinery

Protein subunits: amino acids: L & D isomers

Mirror images of each other

R group = side chains

Aminogroup

Carboxylic acid group

Only L amino acids found in proteins. C chiral, L & D isomers not symmetrical, except glycine

The 20 Amino Acids

The amino acids each have their own shape and charge due to their specific R group.

View the molecular shape of amino acids by clicking on the URL link below:

http://sosnick.uchicago.edu/amino_acids.html

Would the shape of a protein be affected if the wrong amino acid were added to a growing protein chain?

Ionization state as a function of pH

Physiological pH (measure of [H+])

Simplest amino acids

Ball & stick

Stereochemical

Fischer projections

Aliphatic side chains

M: thioether(-S-)

Ile: 2ndchiral center

Aliphaticside chainshydrophobic

Proline: cyclic structure

Ring structure: Proline conformationally restricted, marked effecton protein architecture

Aromatic side chains

Cysteine

Similar to Serine with sulfhydryl, or thiol (-SH) group replacinghydroxyl (-OH) group

-SH more reactive than -OH. -SH pairs form disulfide bonds(aka bridges), key role stabilizing proteins

The basic amino acids

Polar side chains

Lys & Arg havepositive chargesat neutral pH

His can bepositivelychargednearphysiologicalpHLys side chain

capped withamino group

Carboxylate & Carboxamide side chains

pKa of some amino acids

Amino acid abbreviations

16

Essential Amino Acids

• 10 amino acids not synthesized by the body

• arg, his, ile, leu, lys, met, phe, thr, trp, val

• Must obtain from the diet

• All in diary products

• 1 or more missing in grains

and vegetables

Primary structure: Peptide bond, between AAs

Between -carboxyl group of one AA & -amino group of another

2 amino acidsDipeptide

Loss ofH2O

Equilibrium favors hydrolysis, hence,biosynthesis of peptide bonds require free energy input

Peptide bonds are stable kinetically

Polypeptide chain has direction

Main chain or backbone

Constant backbone: regularly repeating part

Distinctive side chains (R-groups): variable part

AA unit in a polypeptide is called a residue, which contains,a carbonyl group; good hydrogen-bond acceptor,an NH group (except Pro); good hydrogen-bond donor

Cross links (disulfide bridges)Prevalent mainly in extracellular proteins

Bovine insulin: AA sequence1953, Fred Sanger determined aa sequence of insulin, landmark!

Showed for 1st time, protein has precisely defined aa sequenceAlso showed that only L-amino acids were present, linked by peptide bonds

Now, aa sequence of > 100,000 proteins are known

1950s-1960s studies showed aa sequence genetically determinedEach of 20 aa encoded by one or more specific sequences of3 nucleotides.

Polypeptide bonds are planar

Six atoms (Ca, C, O, N, H, Ca) lie in a plane, in a pair of aa

Bond lengths in peptide unit

Trans & cis peptides

Cis configuration has steric hindrance; trans strongly favored

Rotation of bonds in a polypeptide

Amino group to C & carbonyl group to C are pure single bonds,allow rotationFreedom of rotation allows proteins to fold in different ways

Dihedral angle: measure of rotation about a bondbetween -180o

& +180o

Ramachandran diagramMost angle combinations (75%) excluded by steric hindranceDark green most favored

Steric exclusion: powerful organizing principle

Limited conformations favor protein folding, favorable entropy of too many conformations opposes folding

Secondary structure: (1) alpha helix1951, predicted by Pauling & Corey, 6 years before it was seen!

ribbon

ball & stick, sideend view

space-fillingcore

alpha helix stabilized by hydrogen bonds

CO group of residue n forms H-bond with NH group of Residue n + 4

Ball & stick model of alpha helix

Ribbon and cylindrical depiction

Residues related toeach other bya rise of 1.5 Å and a rotation of 100degrees.

3.6 aa residues / turn

Pitch = 5.4 Å(1.5x3.6)

Ferritin, an iron storage protein

75% alpha helix

Helical content of proteins ranges widely

Super helix: alpha helical coiled coil

Can be as long as 1000 Å, very stable

Helical cables in these proteins serve a mechanical role,forming stiff bundles of fibers

Found in: • myosin and tropomyosin in muscle,• fibrin in blood clots,• keratin in hair, quills, claws, hoofs, & horns• intermediate filaments (cytoskeleton or internal scaffolding of cells)

Structure of a beta strand

Side chains are alternately above and below plane of backbone

Distance between adjacent aa = 3.5 AContrast to 1.5 A for alpha helix

Also predicted by Pauling & Corey

Antiparallel beta sheet

Strands linked by H-bonding between opposite amino acids

Parallel beta sheetStrands linked by H-bonding of an aa on one strand to twodifferent aa on the adjacent strand

Structure of mixed beta sheet

Fatty acid-binding protein

Rich in beta sheets

Arrow pointingto carboxyl-terminal end

Tertiary structure, myoglobin

O2 carrier inmuscle,

1st protein inatomic detail,

153 aa,

X-ray crystals

Tertiary structure, myoglobin, schematic

Mainly alpha helices,total = 8 helices (75% of main chain)

Prosthetic (helper)group to bind O2

Heme group isprotoporphyrin IX,& central iron atom

Distribution of aa in myoglobin

Yellow: hydrophobic aaBlue: charged aaWhite: other aa

Cross-section

Surface, mainly charged aa. Interior, mainly hydrophobic aa

Quaternary structure, dimerCro protein of bacteriophage lambda

Dimer of identical subunits

Quaternary structure, tetramer

Humanhemoglobin,two alpha(red)two beta(yellow)subunits,

4 heme groupsCovalent bond…..NO

Amino acid sequence determines 3D-structureBovine ribonuclease, 1950, C. Anfinsen work

4 disulfide bonds124 amino acids

Denature &renature

Primary structure determination

•Acid hydrolysis

•Column chromatography

•Ion exchange chromatography

Reducing disulfied bonds

beta-mercaptoethanol, reduced

oxidized

Denaturing agent, urea

Denaturing agent, guanidinium chloride

Denaturing agent, beta mercaptoethanol

Ribonuclease: reduction & denaturation

Finishing touches: covalent modifications

Proteins covalently modified to augment function

Research Protein

Discuss what you can learn about its structure, function and the organism it

comes from using the skills you learned today and website resources.

You can explore a number of proteins using Cn3D. Go to the following URL:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Structure

Thank

You