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8/2/2019 In Vivo Proteon Folding
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MOLECULAR CHAPERONES
AND
IN-VIVO PROTEIN FOLDING
Prepared by-
Bhishma Patel
209BM2246
M.Tech Biotech (1st sem)
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INTRODUCTION
Protein folding is the physical process by which apolypeptide folds into its characteristic andfunctional three dimensional structure from randomcoil
Each amino acid has to some extent a specialcharacter, which determines more or less theposition of the amino acid residue in the nativeprotein, and therefore determines the overall protein
structureC. Anfinsion postulated that all proteins contains
in their primary structure, the complete informationwhich determines the 20 & 30structure
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INVITROVS.INVIVOFOLDING
folding by dilution
in buffer
protein denatured
in a chaotrope
folded
protein
in vitro in vivo
folding
folded
protein
Differences:
1. One has all of the
information
immlediately available
for folding; the other
process is gradual
2. the cellular
environment is very
different (much more
crowded)
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INVITROVS.INVIVOFOLDING
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 anenvironment in which fluctuation in temperature and
chemical composition occurs
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intramolecular
misfolding
X
X
X
X
intermolecular
aggregation
X
X
X
X
X
X
Incorrectmolecular
interactions
&loss of activity
exposed
hydrophobic
residues
CONTD
Non-native proteins expose hydrophobic residues that are
normally buried within the core of the proteinThese hydrophobic amino acids have a strong tendency
to interact with other hydrophobic (apolar) residues -especially under crowding conditions
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MOLECULARCHAPERONES
Molecular chaperones are protein machines thatrecognize non-native states of other proteins and, bycontrolled binding and release, assist these substrateproteins to fold properly.
In the late 1970s, the term Molecular chaperone wascoined to describe the properties of nucleoplasmin:
Nucleoplasmin prevents incorrect interactions betweenhistones 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
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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
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NEEDSOF MOLECULARCHAPERONES
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
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MOLECULARCHAPERONESINVOLVEDIN
IN-VIVOPROTEINFOLDING
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
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Folding pathway for a
model protein
involving three
chaperone
Systems in the bacterial
cytosol
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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.
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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.
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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..
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NASCENTPOLYPEPTIDEASSOCIATED-
COMPLEX (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
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HSP 70 FAMILY
It is very large family of molecular chaperonesinvolved in protein folding, with multiplemembers present in most organism.
e.g. Hsc70
constitutive cytosolic memberHsp70 the stress induced cytosolic form
BiPthe ER form
mHsp70 the mitochondrial form
DnaK prok. Equivalent of Hsc70 foundalso in mitochandria and plastid
Ssa1-4 & Kar2 the homologs of Hsp70 &BiP in yeast respectively.
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CONTD
They have two primary domains:
an ATPase domain
a peptide-binding domain
an ATPase domain consist of four smaller domainsforming 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 hydrophobicresidues, and release them in an ATP-dependent manner
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FUNCTIONOF 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 anHsp70 that facilitates both their translocation and folding
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DNAK REACTIONCYCLE
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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 complexwith substrate proteins
it also binds to some unfolded protein.
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BAG-1
Is an antiapoptotic protein and also interact with several
steroid hormone recepters
It binding to the ATPase domain, stimulate the rate ofATP 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
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Auxilin
100KDa cofactor involved in the Hsp70 mediateduncoating 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 betweenHsp70 and Hsp90
It involved in the refolding of denatured protein in rabbitreticulocyte lysate
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HSP60 FAMILYORCHAPERONIN
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. GroELin prokarotes, mitochondria and
chloroplast
TCP-1 ring complex (TRiC) in eukaryotes
chaperonins was originally coined by Ellis to refer non
heat induced Hsp60
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FUNCTION
GroEL is most studied
It facilitates protein folding by preventing
aggregation and also allow partially folded
intermediates to fold in an environmentconducive 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.
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STRUCTUREOF HSP60/GROEL
It consist of 14 identical subunits in two stackedheptameric 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 againstthermal dissociation.
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HSP60 REACTIONCYCLE
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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
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PREOTEIN DISULFIDE ISOMERASE (PDI)
It is a widely distributed enzyme that catalyzesthe interchange or shuffling of disulfide bondsuntil the bonds of the native conformation areformed.
S-S bond formation occurs rapidly and isfollowed by thiol disulfide rearrangementleading to the correct S-S pairing.
It also binds relatively hydrophobic moleculessuch as steroid and thyroid hormones.
It has two catalytic sites, one near to the NH2terminus and other near to COOH terminus.
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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
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TRIGGER FACTOR
48 kDa protein which was first identified by its ability tomaintain the precursor of a secretory protein in atranslocation 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 beenclearly defined
It binds to nascent cytosolic and secretory polypeptidechain and catalyze protein folding in vitro.
GroEL-TF complex show much greater affinity forpartially folded intermediate than GroEL alone
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SPECIALIZEDCHAPERONES
PapDinvolved in the assembly of bacterial pili.
Hsp47
Found in collagen producing cells
Involved in the folding and processing of procolagen inthe ER.
SecB-
Found in E. coli has two function: it maintain precursorof 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.
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PROTEINFLUXTHROUGHBACTERIAL
CHAPERONESYSTEMS
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MITOCHONDRIALIMPORT/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.
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FOLDINGIN 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.
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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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THANK YOU