STRUCTURE OF PROTEINS

CONTENTS• Overview of Protein Structure• Primary Structure• Peptide bond• Secondary structure• Super-secondary structure• Tertiary and quaternary structure• Unstructured protein• Fibrous protein• Protein folding and disease associated with it• Denaturation of protein

AMINO ACIDS

AMINO ACID, PEPTIDES AND PROTEINS Two amino acid molecules

covalently joined through a substituted amide

linkage peptide Bond

• formed by – removal of the elements of

water• from the α-carboxyl group of

one amino acid • α-amino group of another

When a few amino acids are joined in this fashion, the structure is called an oligopeptide

When many (10 to 50) amino acids are joined, the product is called a polypeptide

molecules referred to as polypeptides generally have molecular weights below 10,000

Those called proteins (having >50 amino acids) have higher molecular weights.

OVERVIEW OF PROTEIN STRUCTURE• Configuration-geometric relationship between a given set

of atoms– Ex- that distinguish L- from D-amino acids

• Conformation – spatial arrangement of atoms in a protein. • Thermodynamically the most stable conformations exist.

• Stabilized Largely by Weak Interactions

• Stability – tendency to maintain a native conformation.

• Unfolded state of protein– high degree of conformational entropy

• Native conformation stabilized by– Disulfide bonds– Noncovalent forces

NONCOVALENT FORCES

◦ Weak (non covalent) interactions Hydrogen bonds Hydrophobic interactions Ionic interactions Van der Walls forces

Noncovalent forces leads to protein folding and contribute to a protein’s stability◦ Cause a polypeptide fold into a unique native conformation◦ Stabilizes the native structure against denaturation

Hydrophobic

Interaction

Forces

Electrostatic Interactions Van der Walls

Forces

Due to properties of water that surround the nonpolar group

◦ ( ionic or salt linkage )

◦ Important in stabilization of protein structure

◦ Binding of charged ligand and substrate

◦ Weakest◦ Van der Walls

contact distance Distance of

maximum favorable interaction between 2 atom

Sum of Van der Walls radii of 2 atoms

HYDROGEN BOND

– When a hydrogen atom covalently bound to and electronegative atom• Donor atom

– is shared with a second electronegative atom• Acceptor atom

– For high bond energy• d= 2.7 – 3.1Å

• Geometrically collinear

H- bondO

H

N

HIERARCHY OF PROTEIN STRUCTURE

PRIMARY STRUCTURE OF PROTIEN Def. – Covalent structure, which includes number and sequence of amino acids.

Importance of primary structure◦ Required to understand

its structure Mechanism of action

◦ Biosynthesis including post-translational modification

◦ Comparison between different animal species – shows essential and non-essential residues.

SEQUENCE COMPARISON• Used to predict the similarity in structure and function of protein. 2

sequences are –– Homologous - when their sequence highly alignable

• Protein that evolved from same gene

– Analogy - structurally similar protein sequence, but no evolutionary relationship found.

– Invariant residue - particular amino acid regularly found at same position.

– Conservative substitution - by another amino acid with similar polarity

– Nonconservative substitution – by another amino acid with different polarity

CLINICAL CORRELATION

• Sickle cell anemia– HbS – a variant of normal hemoglobin

• Nonconservative mutation– 6th position of β-globin gene– Glutamic acid(polar) → Valine(nonpolar)

• So deoxy-HbS molecules get polymerized– Precipitation of Hb within RBC

• Sickle shape and hemolysis– HbC – Lysine is substituted for glutamic acid at

6th position (A3 helix) in β chain– HbD –  β121 (GH4) – Glu → Gln

PROTEOLYTIC CLEAVAGE OF INSULIN Proinsulin –

◦ Produced in pancreatic islet cells.◦ Single polypeptide chain containing

86 amino acids 3 intra-chain cystine disulfide bond

◦ Cleaved by proteases present in islet cells

◦ Releases 2 molecule C-peptide – 35-residue fragments Insulin

Insulin contain◦ 2 polypeptide chain

A – 21 AA B – 30 AA

◦ Covalently joined by the same disulfide bond

DIFFERENT TYPE OF INSULIN USED

Porcine insulin◦ More acceptable than bovine insulin

As sequence is more similar to human insulin Bovine insulin

Human insulin – ◦ Primary insulin in developed country◦ Prepared from

Genetically engineered bacteria Modifying pork insulin

Majority of the population are able to utilize animal insulin without complication◦ Because in amino acid sequence

Small number and conservative nature of change They do not significantly change the 3-dimensional structure

PEPTIDE BOND IS RIGID AND PLANAR X-ray diffraction studies of

crystals of amino acids –◦ peptide C N bond ⎯ –

somewhat shorter than the C N bond in a simple amine⎯

atoms associated with the peptide bond are coplanar

Partial double-bond in character cannot rotate freely

◦ Rotation is permitted about the N C⎯ α and the Cα C ⎯bonds

Three bonds separate sequential α-carbons in a polypeptide chain.

The N C⎯ α and Cα C bonds can rotate.⎯The peptide C N bond is not free to rotate ⎯Other single bonds in the backbone may also be rotationally

hindered◦ Depending on the size and charge of the R groups

Φ(phi) bond – between nitrogen and α-carbon

Ψ(psi) bond – between α-carbon and carbonyl carbon

RAMACHANDRAN PLOT

• Ramachandran plot for L-Ala residues.

• Peptide conformations are defined by the values of Ψ and Φ

• Conformations deemed possible are those that involve little or no steric interference,

• The regions are conformations that are

Color Conformation alloweddark blue

involve no steric overlap

medium blue

extreme limits for unfavorable atomic contacts

lightest blue

permissible if a little flexibilityis allowed

yellow not allowed.

SECONDARY STRUCTURE OF PROTIEN

particularly stable arrangements of amino acid residues giving rise to recurring structural patterns– Local spatial arrangement of its main-chain atoms

– Confifurational relationship between tesidues which are about 3-4 amino acids apart in linear sequence.

Contributed by ◦ each amino acids. ◦ Φ-bond

◦ Ψ-bond

◦α-helix and β-strand conformations – most thermodynamically stable.

REGULAR SECONDARY STRUCTURE

– Occurs in segments of a polypeptide chain in which

– All Φ angles are equal.

– All Ψ angles are equal.

Helical structures are characterized by◦ n – number of residues per

turn of helix◦ d – distance between α-

carbon atoms of adjacent amino acids

◦ pitch – distance between repeating turn of helix on a line drawn parallel to helix axis p = n × d

d

p

Α- HELICAL STRUCTURE

◦ Right-handed.◦ n = 3.6◦ Peptide bond plane –

parallel to the axis of helix.◦ Each peptide form 2

hydrogen bonds peptide bond of 4th residue

above and below◦ Optimum geometry and

distance for maximum hydrogen bond strength.

H-bond

Β- STRUCTURE

– A polypeptide is hydrogen bonded to another polypeptide chain aligned in a parallel or antiparallel direction.

– Hydrogen-bonded β-strands appear like a pleated sheet.

Anti-parallel

Parallel

IMPORTANCE OF SECONDARY STRUCTURE

Structure Characteristics

Examples of occurrence

α-helix, cross-linked by disulfide

bonds

Tough, insoluble protective

structures of varying hardness

and flexibility

Keratin of hair, feathers, and nails

β-Conformation Soft, flexible

filamentsSilk fibroin

SUPER-SECONDARY STRUCTURE

Motif – recognizable folding pattern involving ◦ two or more elements of secondary structure ◦ connection(s) between them

May or may not be independently stable

Any advantageous folding pattern

not a hierarchical structural element falling between secondary and tertiary structure

EXAMPLES OF DIFFERENT MOTIFS

– Helix-turn-helix –• in many DNA-binding

protein.

– Strand-turn-strand –• in protein with antiparallel

β-structure.

– Alternating strand-turn-helix-turn-strand –• in many α/β-proteins

HelixHelix

turn

TERTIARY STRUCTURELocation of each atoms in space. Includes

◦ geometric relationship between distant segments of primary and secondary structure and

◦ Positional relationship of the side chain with one another.

◦ Hydrophobic side chains are generally interior.◦ Ionized side-chains are on the outside

Stabilized by water of solvation.

• (a) The polypeptide backbone in a ribbon.• (b) Surface contour image; this is useful for visualizing pockets

– Where other molecule might bind.• (c) Ribbon representation including side chains • (d) Space-filling model with all amino acid side chains.

– Each atom depicted as size of its van der Waals radius

STRUCTURE OF MYOGLOBINHeme prosthetic group

binds One molecule of oxygen8 α helices, Rest forms turns and

loops due to prolineNo β sheets

Structural domain – a compact globular structural unit formed within the polypeptide with

Hydrophobic core Hydrophilic surface

◦ Typically contain 100 -150 amino acids◦ Domains in multi-domain protein

Connected by a segments that may lack secondary structure

Fold – arrangement of secondary structure elements of a domain.

IMPORTANCE OF TERTIARY STRUCTURE

Trypsin contain◦ 2 domain◦ Cleft in between that contain

Substrate-binding catalytic site

An active site within an interdomain surface is characteristic of many enzyme

In enzyme with more than one substrate or allosteric effector site◦ Different site may be located in different domain

CALMODULIN AS EXAMPLE Calcium ion bind in calmodulin

◦ Within the loop of helix-turn-helix motif Called an EF-hand

Fold of calmodulin domain ◦ Containing 2 EF-hand motifs◦ Interconnected by an α-helical

segment

Addition of side chain group –◦ Generate complete tertiary

structure of domain

QUATERNARY STRUCTUREArrangement of polypeptide chain in multi-chain

protein.◦ Subunits in a quaternary structure are associated

non-covalently.

Quaternary structure of de-oxy hemoglobin.• X-ray diffraction-analysis of

de-oxy hemoglobin• (a) A ribbon

representation. • (b) A surface contour

model. • The α-subunits are shown

in shades of gray• The β-subunits in shades of

blue.• Heme groups (red) are

relatively far apart.

STRUCTURE OF HEMOGLOBIN

• Tetrameric Protein• 2 α Globin Chains and 2

β globin chains held by non-covalent interactions

• Bind 4 molecules of oxygen

• Cooperative binding

UNSTRUCTURED PROTEIN Intrinsically unstructured protein – protein with a non-

folded conformationPartially unfolded conformation

Ex –scaffold proteins, hormones, cyclin-dependant kinase and their inhibitors

Functions –by binding to other protein or to DNA and RNA◦ The property of weak binding often advantageous

NEWER ADVANCES

Protein Complexes ◦ Protein molecules in the

cellular milieu are present in protein complexes containing multiple protein subunits.

◦ The complexes typically have 5-10 proteins.

Network – proteins present in 2 or more different complexes can move between complexes to connect them into network.

• Hub – complex that interconnects with >3 other complexes in the network

– Important target for drug therapies.

• Interactom – functional network comprising interactive protein complexes

Stem cell marker proteins Nanog and Rex1 (green) were used to pull down interacting proteins, including core (blue) and peripheral (red) targets

CLASSIFICATION OF PROTEIN ACCORDING TO HIGHER LEVEL OF STRUCTURE• Globular Protein –

– Spheroidal shape– Vary in size– Relatively high water

solubility.– Function as

• catalyst

• Non-globular protein–• Low water solubility

– Fibrous protein• Larger amount of

regular secondary structure

• Long cylindrical shape– Membranous protein– Lipoprotein– Glycoprotein

COLLAGEN

◦ Family of extracellular proteins present in present in all tissues and organs

◦ Most prominent protein in human.

◦ Provides framework that give tissues form and strength.

Amino acid composition◦ Rich in

Glycine Proline 4-hydroxyproline 5-hydroxylysine

Amino acid sequence◦ In all collagen type there are

region with tripeptides repeats Gly-Pro-Y Gly-X-Hyp

COLLAGEN TRIPLE HELIX

Tropocollagen consists of three fibers◦ Three intertwined polypeptide

strands twist to the left wrap around one another in a

right-handed fashion Highly resistant to unwind

◦ n = 3.3◦ stabilized by hydrogen bonds

between residues◦ Additional stability

covalent cross-links modified lysyl residues

Disease Menkes' syndrome

Ehlers-Danlos syndrome

scurvy

etiology dietary deficiency of the copper

Genetic disease dietary deficiency of vitamin C

defect

required by lysyl oxidase

catalyzes a key step in formation of the covalent cross-links

◦ defects in the genes that encode collagen-

1 procollag

en N-peptidase

lysyl hydroxylase

• prolyl and lysyl hydroxylases

• deficit in the number of hydroxyproline and hydroxylysine residues

• undermines the conformational stability of collagen fibers

Clinical feature kinky hair and

growth retardation

◦ mobile joints

◦ skin abnormalities

bleeding gums

swelling joints poor wound

healing ultimately

death

ELASTIN Gives tissue and organ capacity

to stretch without tearing

Abundant in ◦ Ligaments◦ Lungs◦ Wall of arteries◦ Skin

Unordered coiled structure◦ Amino acid residue within folded

structure highly mobile◦ Allysine form cross-links in

elastin◦ Form the heterocyclic structure

of desmosine or hemidesmosine

KERATIN– in which each polypeptide

is α-helical– Sequences in both proteins

shows tandem repetition of 7 residue segments

– Super twisted Coiled coil– Rich in hydrophobic

residues– Left handed opposite in

sense to α helix• Epidermal layer of skin• Nails• Hair

PROTEIN FOLDING

Polypeptide sequence contain information for spontaneous folding

Folding is under ◦ thermodynamic◦ kinetic control

Initiated by short-range noncovalent interaction.

Partially folded structure intermediate ◦ Interact with each other to form a molten-globule state

CHAPERON PROTEIN

Also called ‘Heat shock protein’

Synthesis increased at high temperature

Prevent protein aggregation prior to completion of folding

Bind to polypeptides shielding the hydrophobic surface

Also required for refolding of protein after they cross cellular membranes

PRION PROTEIN DISEASES

Prion protein◦ Infectious agent in absence of DNA

or RNA

◦ Can occur Spontaneously Inheritance of mutated Prion protein

◦ Clinical features Ataxia Dementia Paralysis Almost always fatal.

deposition of insoluble protein aggregates in neural cells

include ◦ Creutzfeldt–Jakob disease in

humans◦ scrapie in sheep◦ bovine spongiform

encephalopathy (mad cow disease) in cattle

◦ vCJD younger patients

PrPc - highly soluble cellular conformation of prion protein◦ glycoprotein ◦ short arm of chromosome 20◦ 3 α-helical◦ 2 small β-strand◦ rich in α-helix

PrPsc - insoluble toxic conformation◦ Conversion of α-helix → β-strand◦ many hydrophobic aminoacyl side

chains exposed to solvent◦ accumulating PrPsc units coalesce

insoluble protease-resistant aggregates ◦ serve as template for

conformational transformation of PrPc

molecules Many times of its number

BETA-THALASSEMIAS

genetic defects ◦ impair the synthesis of

one of the polypeptide subunits of hemoglobin

α-hemoglobin-stabilizing protein (AHSP) ◦ specific chaperone ◦ binds to free hemoglobin -

subunits awaiting incorporation into

the hemoglobin multimer

absence of this chaperone◦ free α-hemoglobin

subunits aggregate ◦ role for AHSP in

modulating the severity of -thalassemia in human subjects.

ALZHEIMER'S DISEASE

Refolding or misfolding ◦ protein endogenous to human brain

tissue, β-amyloid Main cause remains elusive characteristic senile plaques and

neurofibrillary bundles ◦ aggregates of the protein β-amyloid◦ 4.3-kDa polypeptide ◦ Proteolytic cleavage of a larger

protein amyloid precursor protein

◦ conformational transformation soluble α-helix–rich → rich in β-sheet

◦ prone to self-aggregation

amyloid fibers in Alzheimer's - Crystal structure of a segment from the amyloid-beta protein

Normally the two protein sheets are tightly associated in the spine of the fiber but in this case orange-G has wedged its way between the two sheets. 

DENATURATION OF PROTEIN

• Loss of three-dimensional structure sufficient to cause loss of function – without breakage of any peptide bond– rupture of ionic bond, hydrogen bond and hydrophobic bond.

– ↓ solubility– ↑ precipitability– ↑ digestibility

• COAGULATION– irreversible denaturation

• FLOCCULATION-– Precipitation of proteins at iso-electric pH