MOLECULAR CHAPERONES AND IN-VIVO PROTEIN FOLDING

Prepared byBhishma Patel 209BM2246 M.Tech Biotech (1st sem)

INTRODUCTION
Protein

folding is the physical process by which a polypeptide folds into its characteristic and functional three dimensional structure from random coil Each amino acid has to some extent a special character, which determines more or less the position of the amino acid residue in the native protein, and therefore determines the overall protein structure C. Anfinsion postulated that all proteins contains in their primary structure, the complete information which determines the 20 & 30 structure

IN VITRO VS. IN VIVO FOLDING

in vitro in vivo

protein denatured in a chaotrope Differences: 1. One has all of the information immlediately available for folding; the other process is gradual

folding by dilution in buffer

folding

folded protein

2. the cellular environment is very different (much more crowded)

folded protein

IN VITRO VS. IN VIVO FOLDING

The

environment in which the protein folding occurs within cell. - The concentration of unfolded & nacent chain in the cytoplasm & in the context of ribosomes very high. Formation of tertiary structure requires the presence of complete polypeptide or at least a complete protein domain. After folding, protein must fulfill their function in an environment in which fluctuation in temperature and chemical composition occurs

CONTD
Non-native

proteins expose hydrophobic residues that are normally buried within the core of the protein These hydrophobic amino acids have a strong tendency to interact with other hydrophobic (apolar) residues especially under crowding conditions exposed hydrophobic residues

X X

intramolecular
X X

Incorrect molecular interactions & loss of activity

X

X X

intermolecular
X X X

misfolding

aggregation

MOLECULAR CHAPERONES
Molecular chaperones are protein machines that recognize non-native states of other proteins and, by controlled binding and release, assist these substrate proteins to fold properly. In the late 1970s, the term Molecular chaperone was coined to describe the properties of nucleoplasmin: Nucleoplasmin prevents incorrect interactions between histones and DNA In the late 1980s, the term molecular chaperone was used more broadly by John Ellis to describe the roles of various cellular proteins in protein folding and assembly

Requirements for a protein to be considered a chaperone: (1) interacts with and stabilizes non-native forms of protein(s) - technically also: folded forms that adopt different protein conformations (2) not part of the final assembly of protein(s) means it act as catalysts

NEEDS OF MOLECULAR CHAPERONES

To prevent the misfolding and aggregation during the folding of newly synthasized chains To prevent nonproductive interaction with other cell component To direct the assembly of larger protein and multiprotein complexes During stress condition, help in refolding of denatured proteins Assisting in the process of proteolytic degradation

MOLECULAR CHAPERONES INVOLVED IN IN-VIVO PROTEIN FOLDING

Small Heat Shock Proteins Hsp 40 and DnaJ Family Nascent polypeptide associated-complex (NAC) Hsp 60 Family Hsp 70 Family Hsp 90 Family Hsp100 Family Protein Disulfide Isomerase Peptidyl Prolyl Isomerase / Trigger Factor Specialized chaperones

Folding pathway for a model protein involving three chaperone Systems in the bacterial cytosol

SMALL HEAT SHOCK PROTEINS

Consist of 12-43 KDa proteins that assembles into large multimeric structure. They prevent protein aggregation in an ATP dependent manner and also to some extent solubilize aggregates. Little is known about mechanism of action. It has been suggested that the substrate protein coats the outside of the large chaperone multimer and that hydrophobic interactions are critical in substrate binding e.g -crystallins found in our eye lenses where its major role is to bind denatured protein and prevent their aggregation.

DNAJ/HSP40 FAMILY

It consist of 100 menber, defined by presence of highly conserved J domain of 78 residues. J domain is a motif of 4 -helices with conserved sequence HPD in loop between helix 2&3 which followed by nonconserved C terminus It require for efficient binding of protein to Hsp70 through simulation of its ATPase activity It can directly interact with denatured substrate proteins DnaJ/Hsp40 has been proposed to bind with nascent polypeptides to prevent premature folding and to target Hsp70 to them.

HSP 90 FAMILY
Highly

conserved and essential protein found in all organism from bacteria to human e.g in Euk. Hsp 90, the ER form Grp94 and the E.coli homolog HtpG. It has some specific interaction, e.g. with cytoskeleton elements, signal transduction proteins and protein kinase. In vivo function are poorly understood. It functions in association with other cofactors like, PPI family, FKBP52, p23 and steroid recepter complex consist of Hsp90, Hsp70, p48 etc..

NASCENT POLYPEPTIDE ASSOCIATEDCOMPLEX (NAC)

It

is heterodimer of 21KDa & 33KDa subunit It binds to nascent chain at ribosome exit site It prevent the association of ribosome with protein translocation machinery of the ER membrane. It involved in the targeting pre proteins to different sub cellular location such as ER and mitochondria Also cooperate with the Hsp70 system in preventing early folding and aggregation of protein

HSP 70 FAMILY
It

is very large family of molecular chaperones involved in protein folding, with multiple members present in most organism. e.g. Hsc70 constitutive cytosolic member Hsp70 the stress induced cytosolic form BiP the ER form mHsp70 the mitochondrial form DnaK prok. Equivalent of Hsc70 found also in mitochandria and plastid Ssa1-4 & Kar2 the homologs of Hsp70 & BiP in yeast respectively.

CONTD
They have two primary domains: an ATPase domain a peptide-binding domain an ATPase domain consist of four smaller domains forming two lobes with a deep cleft within which the MgATP & MgADP bind. a peptide-binding domain bind to segments of unfolded polypeptides, particularly those containing hydrophobic residues, and release them in an ATP-dependent manner

FUNCTION OF HSP70/DNAK
DnaK, in cooperation with DnaJ, binds to exposed hydrophobic segments of the nascent polypeptide chain and prevent misfolding or aggregation. Associated with proteins that are translocated into the lumen of the endoplasmic reticulum in a co-translational manner and prevents misfolding or aggregation In the case of mitochondria, unfolded pre-proteins are generally transported post-translationally across both membranes into the matrix, where they interact with an Hsp70 that facilitates both their translocation and folding

DNAK REACTION CYCLE

HSP70 COCHAPERONES

GrpE found in bacteria and mitochondria and facilitates nucleotide release from Hsp70 Detailed mechanism is unclear Hip It stabilise the ADP state of Hsc70 that has a high affinity for substrate protein - forming stable Hsp70 complex with substrate proteins it also binds to some unfolded protein.

BAG-1 Is an antiapoptotic protein and also interact with several steroid hormone recepters It binding to the ATPase domain, stimulate the rate of ATP hydrolysis by increasing the rate of release of ADP from Hsp70 P16 It modulates the Hsc70 function by maintaining Hsc70 in a monomeric state and by dissociating unfolded protein from Hsc70

Auxilin 100KDa cofactor involved in the Hsp70 mediated uncoating of clathrin-coated vesicles. It binds to assembeled clathrin lattices and in the presence of ATP , recruits Hsp70. The presence of J domain at COOH terminus indicates its a member of DnaJ family Hop 60KDa protein that can form physical link between Hsp70 and Hsp90 It involved in the refolding of denatured protein in rabbit reticulocyte lysate

HSP60 FAMILY OR CHAPERONIN

They assemble into large, double ring structures and, together with the co-chaperonin known as Hsp10 or GroES, provide a central cavity that allows proteins of size up to about 6065 kDa to fold in a protected environment. e.g. GroEL in prokarotes, mitochondria and chloroplast TCP-1 ring complex (TRiC) in eukaryotes chaperonins was originally coined by Ellis to refer non heat induced Hsp60

FUNCTION
GroEL

is most studied It facilitates protein folding by preventing aggregation and also allow partially folded intermediates to fold in an environment conducive to stabilizing the native state It also function by unfolding the misfolded state so as to allow their productive folding. Member of Hsp60 family also involved in the assembly of large multiprotein complex.

STRUCTURE OF HSP60/GROEL
It consist of 14 identical subunits in two stacked heptameric rings, each containing central cavity. GroEL subunit consist of three domains: Equatorial contains nucleotides binding site Intermediate binds substrate protein Apical binds GroES

In Euk. Similar complex called TRiC which is hetero oligomer of 8 different subunits. In thermophillic archea, the chaperonin is a homooctamer with build in lid, for stability against thermal dissociation.

HSP60 REACTION CYCLE

HSP100 FAMILY
Contain ATP

and polypeptide binding domains Both Hsp104 and Clp form six membered ring complex No human analogs of Hsp104 have been found It may act in concert with Hsp70 and DnaJ homologs to increase the yields of renatured protein Hsp104 has been observed to solubilize thermally aggregated proteins both in vivo and in vitro

PREOTEIN DISULFIDE ISOMERASE (PDI)

It

is a widely distributed enzyme that catalyzes the interchange or shuffling of disulfide bonds until the bonds of the native conformation are formed. S-S bond formation occurs rapidly and is followed by thiol disulfide rearrangement leading to the correct S-S pairing. It also binds relatively hydrophobic molecules such as steroid and thyroid hormones. It has two catalytic sites, one near to the NH2 terminus and other near to COOH terminus.

PEPTIDYL PROLYL ISOMERASE (PPI)

PPI catalyzes the interconversion of the cis and trans isomers of Pro peptide bonds (Fig. 48b), which can be a slow step in the folding of proteins that contain some Pro residue peptide bonds in the cis conformation. Three unrelated families are known: the cyclophilins FK506-binding protein (FKBP) parvulins

TRIGGER FACTOR
48 kDa protein which was first identified by its ability to maintain the precursor of a secretory protein in a translocation competent form in E.coli Trigger factor has three domains: an amino-terminal ribosome-binding domain a middle domain with prolyl isomerase activity a carboxy-terminal domain with no function has been clearly defined It binds to nascent cytosolic and secretory polypeptide chain and catalyze protein folding in vitro. GroEL-TF complex show much greater affinity for partially folded intermediate than GroEL alone

SPECIALIZED CHAPERONES
PapD involved in the assembly of bacterial pili. Hsp47 Found in collagen producing cells Involved in the folding and processing of procolagen in the ER.

SecBFound in E. coli has two function: it maintain precursor of some exported protein by preventing their aggregation or folding to their native state in cytoplasm and it delivers both nascent and completed precursor to SecA.

PROTEIN FLUX THROUGH BACTERIAL

CHAPERONE SYSTEMS

MITOCHONDRIAL IMPORT/ FOLDING

Molecular chaperones play critical role in targeting protein to the mitochondria and the subsequent folding of the imported protein. Two different mHsp70 complexes The ADP bound form favors formation of a complex on the inner membrane that contains mHsp70, its membrane anchor Tim44 and mGrpE. The ATP bound form favors the frmation of a folding complex in the matrix that contains mHsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE.

FOLDING IN ER
Folding begins with the insertion of a preprotein into the lumen of the ER and can occur either posttranslationally or cotranslationally ER has excellent quilty control mechanism that selectively retain misfolded protein which are either degraded or refolded. Several proteins have been identified which are involved in folding in ER BiP, Hsp90, calreticulin, three member of thioredoxin superfamily: PDI, ERp72 and p50.

CONCLUSION
Molecular chaperones recognize and bind to nascent polypeptide chains and partially folded intermediates of proteins, preventing their aggregation and misfolding. Wide variety of techniques ranging from genetics to biophysics have begun to unravel the complexities of these chaperone machines. Different cellular locatons, with their different role in production of new proteins, have specific chaperone systems tailored to the demand of the specific location.

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