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Protein Folding and Molecular
Chaperones
Contents
Protein Folding
Molecular Chaperones
Protein Folding and Diseases
1. Protein Folding
Protein-Folding Problem
 1958
 John Kendrew et al., published
the first structure of a globular
protein, myoglobin.
 “ Perhaps the most remarkable
features of the molecule are its
complexity and its lack of
symmetry”
 1962
 Nobel prize in Chemistry was
awarded to Max Perutz and
John Kendrew.
 Now
 ~80,000 structures in protein
database (PDB)
Common Structural Patterns
 Motifs, folds, or supersecondary structures
 Stable arrangements of several elements of
secondary structure
 Domains
 Stable, globular units
Classification of Protein Structures
 Structural classification of proteins (SCOP) database
 Classification

 All a
 All b
 a/b : a and b segments are interspersed or alternate
 a + b : a and b regions are segregated
~1,200 different folds or motifs
 Protein family (~4,000)
 Proteins with similarities in
 Primary sequence
 (and/or) Structure
 Function
 Superfamily
 Families with little primary sequence similarity but with
similarities in motifs and function
Structural classification from SCOP
database
Structural classification from SCOP
database
Amino Acid Sequenc Determines
Tertiary Structure
 Amino acid sequence
contains all the information
required to protein folding
 First experimental evidence
by Christian Anfinsen
(1950s)
 Denaturation of ribonuclease
with urea and reducing agent
 Spontaneous refolding to an
active form upon removal of
the denaturing reagents
Protein Folding is not a trialand-error process
 E. coli
 make 100 a.a. protein in 5 sec
 10 possible conformations/ a.a.
 10100 conformations
 10-13 sec for each conformation
 1077 years to test all the conformations
Protein folding problem has not
yet been solved
 The physical folding code
 How is the 3D structure determined by the
physicochemical properties encoded in the
amino acid sequence?
 The folding mechanism
 How can proteins fold so fast even with so
many possible conformations?
 Predicting protein structure using
computers
 Can we devise a computer algorithm to
predict 3D structure from the amino acid
sequnece?
The Physical Code of Protein
Folding
 Weak Interactions
 Hydrogen bond
 Hydrophobic interactions
 Van der Waals interactions
 Electrostatic interactions
 Backbone angle preferences
 Chain entropy
 Large loss of chain entropy upon folding
 Covalent bonding
 Disulfide bonding
The Rate Mechanisms of Protein
Folding
 Models for protein folding
 Hierarchical folding
 From local folding (a helix, b sheets) to entire protein
folding
 Molten globule state model
 Initiation of folding by spontaneous collapse by
hydrophobic interactions
Thermodynamics of Protein Folding
 Free-energy funnel
 Unfolded states
 High entropy and
high free energy
 Folding process
 Decrease in the
number of
conformational
species (entropy)
and free energy
 Semistable folding
intermediates
Computing Protein Structures
 Computer-based protein-structure prediction
competition
 Critical Assessment of protein Structure Prediction
(CASP) in every second summer since 1994
Computing Protein Structures
 Template-modeling (homology modeling,
comparative modeling)
 Structure prediction based on the structure
of a protein with a sequence homology.
 Free modeling (ab initio, de novo modeling)
 Fragment assembly
 PDB search of overlapping fragments of target
proteins
 Assembly of fragments using some scoring
functions
 Successful for short proteins (<100 a.a.)
Computing Protein Structures
2. Molecular Chaperones
Molecular Chaperones
 Proteins facilitating protein folding, transport, and
degradation
AAAA
Misfolding
Folding
Translocation
Folding
Degradation
Nuclear import/export
proteasome
Classes of Molecular Chaperones
 Ribosome-associated chaperones
 Trigger factor (prokaryotes)
 RAC, NAC (eukaryotes)
 Cytosolic chaperones
 Hsp70
 Induced in stressed cells (heat shock protein)
 Binding to hydrophobic regions of unfolded proteins, preventing





aggregation
Cyclic binding and release of proteins by ATP hydrolysis and
cooperation with co-chaperones (Hsp40 etc.)
E. coli: DnaK (Hsp70), DnaJ (Hsp40)
Hsp90
Small Hsps
Chaperonin
 Protein complex providing microenvironments for protein folding
 E. coli : 10~15% protein require GroES (lid) and GroEL
 Eukaryotes: TriC/CCT
 Organelle-specific chaperones (eukaryotes only)
 ER chaperones
 Mitochondrial chaperones
Isomerases in Protein Folding
 Protein disulfide isomerase (PDI)
 Shuffling disulfide bonds
 Peptide prolyl cis-trans isomerase (PPI)
 Interconversion of the cis and trans isomers of Pro
peptide bonds
Co-translational Folding
 Prokaryotes
 Trigger factor
 Cyclic association and dissociation with ribosome
 Binds to hydrophobic regions of newly synthesized
polypeptide chains
 Shields nascent chains from degradation by
proteases
 Improve the yields of correctly folded model
substrates by reducing the speed of folding
 Eukaryotes
 Hsp70 and J-protein–based systems
 Ribosome-associated complex (RAC)
 Heterodimeric nascent polypeptide-associated
complex (NAC).
Trigger Factor
Molecular Chaperones
Protein Folding by DnaK and DnaJ
Chaperonin in Protein Folding
Roles of Hsp70 and Hsp90
AAAA
Misfolding
Folding
Hsp70
Metastable
client proteins
Hsp90
Assembly
Hsp70
Hsp90
Folding
Hsp90
 Prokaryotes
 HtpG
 Yeast
 Hsp82, Hsc82
 Higher eukaryotes
HtpG, Trap-1
N
Other Hsp90s
N
 Hsp90a, Hsp90b (cytosol)
 Grp94 (ER)
 Trap-1 (mitochondria)
M
C
M
Acidic linker
C
Hsp90 Chaperone Network in
Yeast
 Genes interacting with Hsp90


~200 physical interactions
~451 genetic and chemical-genetic interactions
Zhao, R. et al., 2005, Cell
McClellan A.J. et al., 2007, Cell
Hsp90 Chaperone Cycle
Open
ATP-bound
Lid
N
ATP
M
C
Lid open : ADP bound
ADP +
Pi
ATP hydrolysis
Closed
Lid close : ATP bound
Class of Hsp90 Co-chaperones
With TPR
Without TPR
Higher
eukaryotes
Yeast
Function
Hop
Sti1
Adaptor to Hsp70
Cyp40
Cpr6, Cpr7
Peptidyl-prolyl isomerase
FKBP51,
FKBP52
-
Peptidyl-prolyl isomerase
Sgt1
Sgt1
Adaptor for SCF and client proteins
PP5
Ppt1
Phosphatase
Aha1
Aha1, Hch1
Activation of Hsp90 ATPase activity
p23
Sba1
Inhibition of Hsp90 ATPase activity
Cdc37
Cdc37
Adaptor for kinases
Chp-1, Melusin
-
Unknown
TPR Motif
 Protein-protein interaction module
 One TPR motif contains two antiparallel a-helices
 Tandem array of TPR motifs generate a righthanded helical structure

TPR domain in co-chaperones binds to MEEVD
sequence in the Hsp90 C-terminus
Hsp90 MEEVD
PP5 TPR
Regulation of ATPase Activity by
Co-chaperones
Sgt1 (CS domain)
Hsp90 N (O)
ATP
ADP + Pi
Hsp90 N (O)
Cdc37 (125-378)
Hsp90 N (C)
Sba1
Aha1 (1-153)
Protein Folding and Aggregation
Conditions Inducing Protein
Aggregation
 Mutations prone to aggregate
 Huntington’s disease
 Familial forms of Parkinson’s disease and
Alzheimer’s disease
 Defects in protein biogenesis
 Translational errors
 Assembly defects of protein complexes
 Environmental stress conditions
 Heat shock
 Oxidative stress
 Aging
Deposition of Aggregates
 Bacteria
 Inclusion body
 Yeast
 Juxtanuclear quality-control compartment (JUNQ)

 Soluble, misfolded, ubiquitylated proteins
Perivacuolar insoluble protein deposit (IPOD).
 Insoluble, terminally aggregated
 Mammals
 Aggresome
Protein Disaggregation
 Hsp70-Hsp104 (ClpB) bi-chaperone
 Hsp70-J protein
 Transfer aggregates to Hsp104
 Hsp104
 Threading activity to refold aggregate
3. Protein Folding and Diseases
Protein-Folding Diseases
 Amyloidoses
 Diseases caused by formation of insoluble amyloid fibers
Protein-Folding Diseases
 Cystic fibrosis
 Misfolding of cystic fibrosis transmembrane conductance
regulator (CFTR)
 Neurodegenerative diseases
 Alzheimer’s, Parkinson’s, Huntinton’s disease, ALS
 Prion diseases
 Mad cow disease (bovine spongiform encephalopathy, BSE)
 Kuru, Creutsfeldt-Jakob disease in human
 Scrapie in sheep
 Prion : proteinaceous infectious only protein
 PrPSc (scrapie) prion form converts PrPC to PrPSc
Formation of Amyloid Fibers
Amyloid –b peptide
Chaperones as Drug Targets
 Hsf1
 Transcriptional activation of heat shock
proteins
 Activators of Hsf1 as drugs for proteinfolding diseases
 Hsp70
 Hsp90
 Clients proteins include some oncoproteins
 Hsp90 inhibitors as cancer drugs
Hsp90 Client Proteins in Cancer
Hsp90 client protein
Roles of Hsp90 client proteins in
cancer
Her2, Raf-1, Akt
Self-sufficiency in growth signals
Plk, Wee1, Myt1
Insensitivity to antigrowth signals
RIP, Akt
Evasion of apoptosis
hTERT
Limitless replicative potential
Hif-1a, Fak, Akt
Sustained angiogenesis
Met
Tissue invasion and metastasis
Hsp90 Inhibitors as Anti-Cancer Drug
Her2,Raf-1,Akt, Hif-1a,
survivin, mutant p53
Oncoprotein
Protein
stabilization
Oncoprotein
Cancer
Hsp90
inhibitor
Bended Form of ATP & 17-DMAG
in the Pocket
J.M. Jez et al, 2003, Chemistry & Biology
Hsp90 Inhibitors
Geldanamycin
ATP
Radicicol
PU3
17-AAG