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Protein Folding in the Cell 2
BIOC 212
Winter 2013
J ason C. Young
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Which amino acid side chains can form these interactions?
can the polypeptide backbone form any of these interactions?
hydrophobic, hydrogen bonds, Van der Waals
hydrophobic interactionsA, G, I, L, V, P, F, W, Y, M,
C, Q, N, T, R, K, E
hydrogen bondsD, E, R, K, H, N, Q, S, T, Y,C
Van der Waals interactions all
ionic bonds D, E, R, K, H
disulfide bonds C
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Protein Folding
Folding is thermodynamically favoured (negative G free energy)
Folding can be spontaneous in principle, but assisted by different
biological mechanisms
Native structure is determined by the primary sequence
Christian AnfinsenNobel 1972
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The Folding Process
Unfolded (denatured) domains have extended conformations with nosecondary or tertiary structure
Folding proceeds through intermediates that have increasingstructure, to the native state
Molten Globule intermediates are close to native
contain some secondary structure elements loosely packed and flexible compared to native state
Folding model of a single-domain protein.Daggett & Fersht (2003) Nature Reviews Mol Cell Biol 4, 497-502.
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Native State
Structure is stabilized by hydrophobic contacts (blue)
Some domains also require a ligand partner to be stable
cofactor (Haem, steroid, etc), or another protein subunit
Some domains are stable without ligand, but can
still bind ligand reversibly
Native state can be in equilibrium with near-nativeintermediates molten globule states
cytochrome B562 with Haem
METLNDTLKVVEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSP
EMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYHQKYR
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Folding Intermediates
Have some secondary structure, but tertiary structure incomplete
Some but not all hydrophobic side chains are buried
More of the polypeptide in flexible, disordered conformation
Risk of aggregation interaction between different unfolded proteinsleads to insolubility
Incompletely folded proteins :
immediately after synthesis
ligand not available
previously native but unfolded by stress (eg. heat)
apo cytochrome B562 without Haem
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Folding Quality Control
Some folding intermediates canbe non-productive off-pathway
Cells have quality controlmechanisms to correct misfolding
Molecular Chaperones assist thefolding of proteins
Proteasomes degrade misfoldedor unfolded proteins
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Chaperones
Molecular Chaperones assist folding and prevent aggregation,without being part of the native state
Chaperones often recognize exposed hydrophobic patches offolding intermediates
Many chaperones are Heat Shock Proteins (HSP; eg. Hsp70 is the70 kDa HSP), highly expressed after stress
Chaperones are also essential under non-stress conditions
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Types of Chaperones
Some substrates require specific chaperones, or combinations ofchaperones
ATP-dependent chaperones actively promote folding
substrate binding and release are regulated by ATPase cycles
ATP-independent chaperones prevent aggregation and can catalyzesome folding steps
Cooperation between chaperones
cytosol endoplasmic reticulum
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Families of Chaperones
Different families of chaperone proteins use various biochemicalmechanisms protein folding toolkit
3 families of ATP-dependent chaperones, with different structuresand ATPase cycles
Chaperonins (Hsp60) Hsp70 Family Hsp90 Family
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Chaperone Families by Compartment
Co-evolution of chaperones with substrates
Chaperonins
(HSP60)HSP70 HSP90 small HSP prefoldin
calreticulin& calnexin
peptidyl-prolyl
isomerase
bacteria + + + + +
archaea + +
eukaryotes(human):
cytosol + + + + + +
ER lumen + + + +
mitochondria + + + +
ATP-dependent ATP-independent
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P
Rotation around backbone is slowed by large side chains
Prolines rotate around backbone the slowest, because of extra
covalent bond
Slow proline rotation may limit the rate of folding
Peptidyl-prolyl isomerases (PPIases) increase proline rotation and
can speed up folding ATP-independent
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HSP70 Family
HSP70 chaperones are 70 kDa monomers
Highly conserved between species, and intracellular compartments
Can have multiple functions in cells in addition to folding
Bertelsen et al. (2009) Proc Nat Acad Sci USA 106, 8471-8476
T. thermophilus Hsp70
ATPase domain
peptide binding domain
ATP peptide
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HSP70 States
ATP-bound the ATPase domain pulls peptide binding domainopen, no substrate peptide binding
ADP-bound the peptide binding domain is separate and shut, fortight substrate binding
two-state mechanism is highly conserved
ATP-bound ADP-bound /nucleotide-free
Kityk et al., Mol Cell 2012 Nov 1 Epub Bertelsen et al., PNAS (2009) 106, 8471-8476
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HSP70 Substrate Binding
Clamp onto short (7 amino acid) polypeptide segments that arehydrophobic and have an extended conformation
There can be more than one Hsp70 binding sites in one polypeptide Many different unfolded proteins can be bound by Hsp70
DnaKpeptide bindingdomain
PeptideNRLLLTG
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DNAJ (HSP40) Co-chaperones
Co-chaperones are proteins which regulate chaperones
DNAJ proteins have a J domain:
J domains bind transiently to Hsp70, activate it to hydrolyze ATPand bind polypeptide
J domains do not bind substrate
Some DNAJ s also have separate substrate binding domains act as ATP-independent chaperones
J domain
Hsc70 ATPasedomain
J iang et al. (2007) Mol Cell 28, 422-433
contact site
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Types of DNAJ s
Type 1 DNAJ s have conserved substrate binding domains
Type 2 have divergent substrate binding domains
Type 3 do not bind substrate directly
activate HSP70s for specialized functions
Type 1 and some Type 2 act in folding
J domain proteins inyeast S. cerevisiae
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Type 1 DNAJ proteins have conservedarrangement of domains
J domain, substrate binding, dimerization
homodimers: 2 x 50 kDa subunits
bind short hydrophobic sequences
Type 1 DNAJ
substratebinding dimerizationJ domain
Type 1 DNAJ co-chaperonesyeast Ydj1 (Type 1)
Ramos et al. J Mol Biol (2008) 383, 155-166
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HSP70 NEFs
Nucleotide Exchange Factors (NEF) replace ADP with ATP andcause release of bound polypeptide by Hsp70
In eukaryotes, there are 3 families of exchange factors: BAG, HspBP1 and HSP110
All open up the ATPase domain to release nucleotide
ATPasedomain
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HSP70 Cycle
DNAJ binds substrate
J domain stimulates ATP hydrolysis by HSP70
substrate is transferred and bound by HSP70 in ADP state
NEFs cause ATP re-binding and release of substrate
Hartl & Hayer-Hartl (2009) Nature Struc Mol Biol 16, 574-581
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HSP70 Proteins
human Hsc70 is constitutively expressed, Hsp70 is inducible
mechanisms are the same
also have forms in human mitochondria, ER lumen
human Type 1 HSP40: DnaJ A1, DnaJ A2
compartment HSP70DNAJ co-
chaperoneNEF
E. coli cytoplasm DnaK DnaJ GrpE
human cytosol Hsc70, Hsp70
DnaJ A1,DnaJ A2,
Hsp40& many others
Bag1, Bag2,
Hsp110& others
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How Does HSP70 Help Folding?
A DNAJ is always required, importance of NEF varies betweenHSP70s and substrates
Multiple, fast cycles of substrate binding and release Hsp70 binding prevents aggregation of a substrate, while release
provides chances for it to fold
Importance of the DNAJ ? hypothesis: 2-point binding of substrate by DnaJ A2 and Hsc70
Type 1 DNAJ
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Names
An atheist, a priest and a rabbi go into a bar.
(categories and their properties are important)
Alice, Bob and Carl go into a bar.
(names are less meaningful unless linked to categories)
What is Alice?
(a woman? an atheist? an alcoholic?)
Alice is an atheist, Bob is a priest and Carl is a rabbi. They all go into abar. How is Alice different from Bob and Carl? What will happen in
the bar?
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HSP70 Cycle
HSP70-ATP HSP70-ADP
Substrate Binding:
HSP70
DNAJ
Co-chaperoneinteraction with HSP70
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End of 2
Hartl et al. (2011) Molecular chaperones in protein folding andproteostasis. Nature 475, 324-332.