Download MBBS Biochemistry PPT 82 Protein Folding Lecture Notes

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Protein Folding

Specific learning objectives

1. Proteostasis pathways

2. Denaturation and renaturation of proteins

3. Steps of Protein Folding

4. Assisted Protein Folding by Chaperons

5. Enzymes involved in Folding Pathways

6. Protein Misfolding and related Diseases
Introduction

Newly synthesized polypeptide folds into its characteristic and

functional 3-D structure via a physical process known as protein

folding.

Interactions among aa lead to formation of a folded 3-D structure

known as native protein which is stable.

Proteins must maintain conformational flexibility to function

Continual maintenance of active set of cellular proteins required under

given set of conditions is called proteostasis

Proteostasis pathways

Fig.4.25. Lehninger Principles of Biochemistry, 6th Ed.
Denaturation at protein levels

At primary structure: Sequence of aa held together by covalent

peptide bonds, is not disrupted by denaturation.

At secondary structure: Proteins lose all regular repeating patterns

such as -helices and -pleated sheets and adopt a random coil

shape.

Cont--

At tertiary structure: Disruption of covalent interactions between aa side

chains (such as disulfide-bridges bet cysteine groups), non-covalent

interactions between polar aa side-chains, van-der waals interactions

between non-polar aa side chains.

At quaternary structure: Protein sub-units are dissociated and/or spatial

arrangement of protein subunits is disrupted.
Loss of protein structure results in loss of function

Native Protein denaturate in presence of reducing agent, alter

pH, temp, ionic strength, and solubility.

Various denaturants are:
1. Heat disrupts hydrogen bonds and hydrophobic interactions bet

non-polar residues. Ex. Albumin in egg denature and coagulate

during cooking.

2. Strong acids and bases disrupts salt bridges formed in a protein

structure. Ex. In digestive system, acidic gastric juices causes

coagulation of milk by proteolytic enzyme renin.

Cont--

3. Reducing agents like guanidine hydrochloride (GdnHCl) or -

mercaptoethanol reduces disulphide bonds to sulfhydryl group and

breaks intra and interchain disulphide bonds

4. Organic solvents, urea and detergents (SDS) disrupts hydrophobic

interactions that stabilize core of globular proteins
? Urea disrupts stabilizing hydrophobic interactions, thus freeing entire

polypeptide from its folded conformation.

5. Extremes of pH alter net charge on protein, causing electrostatic

repulsion and disruption of hydrogen bonding
Amino acid sequence determines tertiary structure

3? structure of globular protein determined by its aa sequence. Proof

of this came from experiments showed that denaturation of some

proteins is reversible

Certain globular proteins denatured by denaturing reagents wil

regain their stable native structure and biological activity. This

process is called renaturation.

Denaturation and renaturation of ribonuclease A, demonstrated by

Christian B Anfinsen got Nobel prize in chemistry 1972

Cont--

He provided first evidence that amino acid sequence of a polypeptide

chain contains all information required to fold chain into its native, 3-D

structure.

3-D structure and function of proteins destroyed by denaturation, which

demonstrate relationship between structure and function.

Some denatured proteins can renature spontaneously to form

biologically active protein.
Renaturation of unfolded, denatured Ribonuclease

Lehninger Principles of Biochemistry by David L Nelson

Protein folding in sequential manner

1. Newly synthesized polypeptide

chain emerges from ribosome,

short segments fold into

secondary structural units

(Image by MIT Open Course Ware, adapted from image by Professor Jonathan

King)
Cont--

2. Folding proceeds via an initial clustering among side chains of

hydrophobic residue which prefer to be aloof from an aqueous

environment:

Clustering due to non-specific interaction among hydrophobic residues

lead to formation of a compact arrangement (molten globule state)

Hydrophobic residues of proteins gather inside collapsed forms within

core

Collapsed state favors formation of 2? structure & encourages 3?

interaction among residues

Assisted Protein Folding

Most Proteins fold spontaneously to their native but some proteins

undergo assisted folding

Folding of some proteins require chaperons, these are specialized

proteins which interact with partially folded or improper folded

polypeptides, ensure correct folding pathways. Ex. Chaperones i.e, Hsp

70 family and chaperonins

Finally, folding pathways of some proteins require two enzymes

(Protein disulfide isomerase and Peptide prolyl cis-trans isomerase)
Cont--

Chaperones: Hsp70 family of chaperones more abundant in cells

stressed by elevated temperatures:

? It binds to regions of unfolded polypeptides rich in hydrophobic

residues may break up protein aggregate or to prevent formation of

new aggregate

? It protect both proteins by heat denaturation
? It blocks folding of certain proteins that remains unfolded until they

translocated across a membrane

? It assisted folding and assembly

Cont--

2. Chaperonins: Chaperones provide microenvironments in which

folding occur:

? They are elaborate protein complexes required for folding of some

cellular proteins that do not fold spontaneously

? Hsp60 acts as chaperonins in folding process, together with an

Hsp70 chaperone

? Central cavity of Hsp60 chaperone provides a sheltered environment

in which a polypeptide fold until all hydrophobic regions are buried in

its interior, thus prevent protein aggregation.
Cont--

Two enzymes involved in Protein Folding Pathways

1) Protein disulfide isomerase (PDI):
? Catalyzes interchange, shuffling of disulfide bonds until bonds

of native conformation are formed

? Catalyzes elimination of folding intermediates with inappropriate

disulfide cross-links

Cont--

2) Peptide prolyl cis-trans isomerase (PPI):

? Catalyzes interconversion of cis and trans isomers of pro residue

peptide bonds, which can be slow step in protein folding that contains

some pro peptide bonds in cis conformation
Chaperone-assisted protein folding

Fig.8.23: The Cel , 4th Ed

Protein Misfolding and Diseases

Incompletely and incorrectly folded proteins leads two serious problems

to cells:

1. Loss of function due to absence of correctly folded protein:
? Cystic fibrosis

2. Aggregation of incorrectly folded proteins:
? Alzheimer's disease (Amyloid beta)
Cystic Fibrosis (CFTR)

Cystic fibrosis transmembrane conductance regulator (CFTR) is a

membrane protein and encoded by CFTR gene.

CFTR gene codes for an ABC (ATP binding cassette) transporter-

class ion channel protein that conducts chloride ions cross epithelial

cell membranes.

Mutations of CFTR gene affect chloride ion channel function leads to

dysregulation of epithelial fluid transport in lung, pancreas resulting in

cystic fibrosis.

Cont--

Caused by deletion of a 3 nucleotides which results in a loss of aa

(Phe) residue at 508th position, causes improper protein folding.

Improved understanding of protein folding may lead to new therapies

for CFTR.
Alzheimer's disease (Amyloid beta)

Refolding or misfolding of -amyloid protein endogenous to human

brain tissue.

Senile plaques and neurofibril ary bundles contain aggregates of

protein -amyloid.

A 4.3 KDa polypeptide produced by proteolytic cleavage of a larger

protein known as amyloid precursor protein (APP).

Cont--

Levels of -amyloid become elevated

This protein undergoes a conformational transformation from a

soluble -helix to -sheet and prone to self-aggregation.
Summary

In protein folding steps, regions of secondary structure may form,

followed by folding into supersecondary structures.

Large ensembles of folding intermediates are rapidly brought to a

single native conformation.

For many proteins, folding is facilitated by Hsp70 chaperones and by

chaperonins.

Cont--

The 3-D structure and the function of proteins destroyed by

denaturation, which demonstrate the relationship between structure

and function.

Some denatured proteins can renature spontaneously to form

biologically active protein.

Disulfide bond formation and cis-trans isomerization of Pro peptide

bonds are catalyzed by specific enzymes.
27

Reference Books

1) Harper's Il ustrated Biochemistry-30th Ed
2) Biochemistry 7th Ed by Jeremy M. Berg, John L. Tymoczko and

Lubert Stryer

3) Lehninger Principles of Biochemistry, 6th Ed.

Thank you

This post was last modified on 05 April 2022