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BMB170a_2011_LECTURE5 - Linderstrøm-Lang D2 O XH OH X XD...

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Unformatted text preview: Linderstrøm-Lang D2 O - XH OH X- XD + ODH2 O – get this rate from model peptides XHburied XHopen for most highly buried groups, this is a measure of Kfolding kpeptide XD exchange – – – – Protein Design - the next frontier PNAS (2001) 98:14274 Science (2003) 302:1364 In vivo folding – – – – – – – – – – – – – – – Molecular chaperones A group of unrelated classes of proteins – Bind to and stabilize an unstable conformation – Facilitates correct fate in vivo – Aren’t part of the final structure Possible correct fates – Folding, oligomeric assembly, transport to organelle, disposal by degradation Molecular chaperones don’t violate Anfinsen’s self assembly principle – No inherent information in chaperone about proteins final fold – Prevent incorrect interactions within and between non-native proteins – Assist self-assembly – Increase the yield but not the rate (except for isomerases) Families of Chaperones – – Cytoplasmic Chaperone Summary Hartl 2002 Science BiP/Hsp70 class Hsp70 family of proteins Location Chaperone Roles Prokaryotic cytosol DnaK cofactors DnaJ, GrpE Stabilizes newly synthesized polypeptides and preserves folding competence; reactivates heat-denatured proteins; controls heat-shock response Eukaryotic cytosol SSA1, SSB1(yeast) Hsc/hsp70, hsp40 (mammalian) Protein transport across organelle membranes; binds nascent polypeptides; dissociates clathrin from coated vesicles; promotes lysosomal degradation of cytosolic proteins ER KAR2, BiP/Grp78 Protein translocation into ER Mitochondria/ Chloroplasts SSC1 ctHsp70 Protein translocation into mitochondria; Insertion of light-harvesting complex into thylakoid membrane Cycle Hsp70s are extremely slow ATPases (>0.003 s-1) Co-chaperones regulate turnover – J-proteins (Hsp40) stimulate hydrolysis ~7-fold – NEFs – increase turnover Nucleotide binding domain Hsc70 NBD (3hsc) Flaherty..McKay (1990) Nature 346:623 DnaK NBD/GrpE (1dkg) Harrison..Hartl, Kuriyan (1997) Science 276:431 Full model Bertelsen..Gestwicki (2009) PNAS (2kho) Hsp70 summary Review: Hartl (2002) Science 295:1852 NEF structures GrpE family – bacterial cytosol/ mitochondria BAG family in eukaryote cytosol HspB1 – eukaryote/ ER Hsp70 related proteins (110/170 in the cytosol) Sondermann et al (2001) Science Prefoldin, a functional substitute for Hsp70 chaperones Minimal chaperone machinery in bacteria – nascent chain binding chaperones (trigger factor and Hsp70) – Group I chaperonin system GroEL/GroES (acts post-translationally) Archaea have thermosome (double ring structure like GroEL/GroES) but most lack Hsp70 equivalent Instead of Hsp70, Archaea use prefoldin (absent in bacteria but found in eukaryotes) to stabilize non-native proteins for subsequent folding in the cavity of a chaperonin Structure of prefoldin Found in eukaryotes and archae Can substitute for Hsp70 2 proteins (ratio 2 :4 ) Hexameric complex (jellyfish?) Six tentacles (rods of - helical coiled coils) pointing away from two 8stranded anti-parallel -barrels Hydrophobic patches on distal regions point to central cavity Flexible tentacles – to adjust to different sized substrates – nascent chains and partially folded proteins Multivalent binding to substrate – detergent-like solubilization of non-native polypeptides prone to aggregation Substrates may protrude into central cavity – shields aggregation-prone states from bulk solution Substrate binding may cause unwinding of coiled coils, exposing more hydrophobic regions Binding to individual tentacles – may be weak and reversible – allows a strong interaction when all six are used – can release to downstream chaperones without ATP hydrolysis. – – – Hsp60 family of proteins Location Chaperone Roles Prokaryotic cytosol GroEL/ GroES Protein folding, including elongation factor, RNA polymerase. Required for phage assembly Mitochondria/ Chloroplasts Hsp60/10 Cpn60/10 Folding and assembly of imported proteins Archaebacterial cytosol TF55 Thermosome Binds heat-denatured proteins and prevents aggregation Eukaryotic cytosol TCP-1, CCT, or TRiC Folding of actin and tubulin; folds firefly luciferase in vitro Chaperonins GroEL/Hsp60 family – – – – – First structure of GroEL GroEL is in bacterial cytoplasm – Two stacked rings Seven 60 kD subunits each (547 aa) Arranged with 7-fold symmetry – Cylinder with central cavity – Rings arranged back-to-back forming interface across equatorial plane In vitro studies – polypeptides diluted from denaturant show that non-native intermediates bind to GroEL with 1-2 polypeptides per 14-mer – Part of bound polypeptide is in central cavity (EM studies) GroES (10 kD) binds at one end of cylinder Diameter of channel: – ~45 Å – Length: ~146 Å – Total volume: ~250,000 Å3 Space consideration – ~100 kD (folded) or 50-60 kD (molten globule) – calculation assumes protein spans the channels of both doughnuts – not big enough (?) Suggested function of GroEL: Anfinsen cage for protein to be isolated while folding. But residues affecting binding are hydrophobic -how does unfolded protein fall off? Group I Chaperonin Mayer (2010) Mol Cell The GroEL-GroES reaction cycle Unfolded protein binds in top ring too big for channel ATP binding – allows GroES release from other side – it (or another GroES) binds to ring with chain – cavity is bigger/hydrophilic - folding starts ATP hydrolysis – weakens interaction between GroES and GroEL ATP binds to opposite ring – release of GroES – release of polypeptide from other ring (~15s) Proteome analysis of Chaperonedependence E. Coli ~2400 soluble proteins in proteome ~250 interact with GroEL (~400 in E. coli lacking TF, DnaJ and DnaK) ~85 proteins require GroEL (use ~80% GroEL) Kerner..Hartl (2005) Cell 122:209 Hsp90 Can prevent aggregation of proteins Interact with ‘clients’ – Transcription factors and protein kinases – Roles in signal transduction, chromatin remodeling, cellular trafficking etc Late stage in folding Can target for degradation Related to DNA gyrase 2iop Hsp90 Cycle Lot’s of structures in different conformations from a variety of methods. General path toward unified model. Mayer (2010) Mol Cell Hop – Hsp Organizing Protein Contains three TPR domains Carboxylate clamp TPR1 binds C-terminus of Hsp70 TPR2A binds Hsp90 Hands off from Hsp70 to Hsp90 Scheufler et al (2000) Cell (1elw) Other examples include protein phosphatase 5 (Ppp5) which binds and phosphorylates Hsp90 or CHIP which targets Hsp70/90 for degradation. Chaperone receptors Review: Kriechbaumer et al (2011) Protoplasma ...
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