Anuradha Bhardwaj and Varij Nayan

Protein Folding

Primary Structure

Tertiary Structure

IN VITRO PROTEIN FOLDING IN VIVO PROTEIN FOLDING

In Vitro
Small chemically denatured proteins have been used High dilution Low temperature Spontaneous folding Completely synthesized proteins are used

In Vivo
Large protein molecules are there Highly crowded environment Dynamic physiological conditions High degree of compartmentalization N-terminus is synthesized before Cterminus Large molecules tend to form aggregates Molecular chaperones needed

What are Molecular Chaperones

Molecular chaperones are proteins which can recognize and bind to non-native (denatured, folding intermediate or molten globule like) polypeptides to facilitate them to fold to their native states that are specified by the primary sequences.

Helper proteins Are not components of the final functional structures. Do not contribute conformational information to the folding process

Nascent Polypeptide

~50% proteins go through simultaneous folding

~20% proteins undergo degradation

~20% proteins bound with chaperones, 30%-40% proteins bound with chaperons at elevated temperature

Two types of Molecular Chaperones:1- Proteins that suppress polypeptides aggregation and

promote protein foldingHeat shock proteins 2- Proteins that promotes correct disulfide bond formationA- Enzymes that catalyze disulfide bond formation (DsbA, DsbB) B- Disulfide bond isomerases that correct the mis-connected disulfide bonds (DsbC, DsbD, Protein Disulfide Isomerase(PDI)

ATP Dependent Chaperones Hsp 70 Hsp 90 Chaperonines

ATP Independent Chaperones Prefoldin

Major Hsp Families

Chaperone Hsp 100(ClpB) Hsp 90 Hsp 70 Function ATP dependent disaggregation and unfolding for degradation. Conformational maturation of steroid hormone receptors. ATP dependent stabilization of hydrophobic regions of extended polypeptides & folding. ATP dependent protein folding. Stabilization against aggregation during heat shock.

Hsp 60 Small Hsps

Hsp 70 70 KDa. Highly conserved family of proteins. Distributed ubiquitously in all prokaryotes & in cellular components of eukaryotic organisms. Binding & release of substrate takes place by cycles of ATP binding & hydrolysis.

Hsp40
40 KDa As a regulator of Hsp70, the J domain of Hsp40 can stimulate the ATPase activity of Hsp70 in order to fold nonnative polypeptides. Hsp40 can bind non-native polypeptides directly and deliver them to Hsp70 for folding.

Domain structure of Hsp70 and Hsp40

ATPase domain
Hsp70 Groove J domain Hsp40 Zinc fingers Lid

Peptide binding domain

Peptide binding domain

Recognition of substrates by Hsp 70

Binding site occurs, on average, every 40 residues. The

peptide binding site of Hsp 70 contains a cleft that binds the

peptide in an extended conformation. This chaperone recognizes linear polypeptide sequences enriched in

hydrophobic amino-acids Hsp 70 prefers to bind the peptide with a minimal length of

seven aminoacids with sequence of XXLLLXX. Structural

studies revealed that selectivity may come from Leu at position 4

DnaKBacterial homolog of Hsp70 Binds to nascent chains

Denovo folding & recovery from stress

Require DnaJ & GrpE

Dna J 41 KDa bind to DnaK & stimulate its ATPase activity, generating the ADP bound state of DnaK, which interacts stably with

the polypeptide substrate.

J-Domain a scaffold of 4 -helices &a solvent accessible loop region that exposes a conserved tripeptide (His-ProAsp) essential for interaction of the J-domain with Hsp70.

Grp E 23 KDa Bind to ATPase domain of DnaK and by distorting the nucleotide binding pocket, induces release of bound ADP

Rebinding of ATP trigger dissociation of the DnaKsubstrate complex Restricted to prokaryotes only

ATP J

Pi

ATP e
ATP

ADP

e
Peptide release

e
ADP

Conclusions of Hsp70 system

Hsp70 prefers to bind hydrophobic sidechains (Leu) of the

peptides in the extended conformations.

ATP hydrolysis in the ATPase domain of Hsp70 may generate conformational changes in the peptide-binding domain to trap the non-native polypeptides for folding. The J domain of Hsp40 can stimulate the ATPase activity of

Hsp70 while the peptide-binding fragment of Hsp40 may

stretch out the non-native polypeptides and present them to Hsp70.

Chaperone Family

Bacterial

Yeast

Mammalian

Hsp 70 70-kDa ATPases, bind extended polypeptides enriched in hydrophobic amino acids

DnaK: de novo folding SSa1-4: de novo Hsc 70: constitutive, and recovery from stress, folding and recovery binds nascent chains binds nascent chains from stress Hsc 70: stress inducib SSb1-Ssb2: blind ribosomes nascent chains Pdr13p/Ssz: binds ribosomes DnaJ: has chaperone Ydj1: has chaperone Hsp40 activity, interacts with activity, interacts with 40-kDa cofactors that DnaK Ssa1 stabilize Hsp 70 substrate Sis1: ribosomeinteractions by stimulating associated, has ATP hydrolysis chaperone activity, interacts with Ssa1 Zuotin: ribosomeassociated, interacts with Pdr13p/Ssz Nucleotide Exchange GrpE: 23-kDa Factors Hsp70- nucleotide exchange factor, interacts with prokaryotic Hsp70 Stil Hop, p60

Bag homologs

Bag1-Bag5

ADP-stabilizing factor

Hip: binds newly translated proteins, stabilizes Hsp 70-ADP complex Trigger factor: ribosomes- associated, binds nascent chains, has proly1 isomerase GimC/prefoldin: binds activity GimC/prefoldin: mechanism unknown, to nascent chains possibly TRiC cofactor; 6 subunits Gim1-Gim6

Hsp70-like chaperones Stabilize extended polypeptides; prevent aggregation

Chaperonin/Hsp60 Barrel-like structures that provide a protected folding environment for substrates. Promote ATP-dependent folding in the chamber of the double-ring complex

GroEL/Hsp60: 800kDa, 14-mer complex, de novo folding and recovery from stress GroEL/Hsp10: 7-mer ring complex, associates with GroEL, acts as a lid for the folding chamber.

TRiC/CCT: 1000 kDa, 16-mer hetro-oligomeric ring complex, has builtin lid; 8 subunits Cct1Cct8

TRiC/CCT: same as yeast complex, assists de novo folding, binds nascent chains

Small \ Holding Chaperones

Prolyl-Isomerase Trigger Factor

Gim Complex \ Prefoldin - ~90 KDa complex of 2 & 4 subunits - actin and tubulin folding

The Chaperonins Ring shaped Chaperones

Hsp 60
Large cylindrical complexes consisting of 2 stacked rings Usually each ring has 7 subunits ATP binding & hydrolysis

Types of ChaperoninsGroup 1
- GroEL/Hsp60 - GroES/Hsp10

Group 2
- TRiC/CCT
- Thermosome Complex

Gro EL Contains 14 identical subunits arranged in two stacked rings of 7 subunits each. Each subunit contains 2 discrete domains joined by a hinge like intermediate domain.

- Equatorial domain - Apical domain

Gro ES A ring shaped complex composed of 7 identical subunits Promotes ATP hydrolysis.

N 7 ATP + GroES 7 ATP - 15 Sec ATP ATP ADP ADP ATP ATP

7 Pi

7 ADP, GroES

Conclusions of Hsp60
GroEL-GroES complex provides a privileged environment in

favor of protein folding

The ATP/GroES binding on the cis ring triggers the folding process. The ATP binding on the trans ring discharges the peptide and GroES. The ATP hydrolysis is used to drive the system to next stage directionally

The hydrophobic residues on the surface of apical domain

facing the central cavity are responsible for peptide substrate binding

Hsp90
ATP binding and hydrolysis dependent.

In eukaryotic cells highly abundant - ~2% of cytosolic proteins essential for viability Hsp90 works in context of a complex assembly that has been termed the foldosome.

Participates in the conformational regulation of signal

transduction molecules -eg.-tyrosine kinase and steroid hormone receptors.

Interaction between Chaperones

Sequential and processive model - the newly translated polypeptide is released into the bulk cytosol only after it adopts a conformation that is committed to fold. Partitioning model - Chaperones interact with substrate proteins in a manner that non-native folding intermediates partition freely through the cytosol, cycling between a network of available chaperones.