Lectures 6 7 -Protein_Folding

Each side can accommodate one unfolded protein 1

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– Each side can accommodate one unfolded protein • 1 GroES binds to one side of GroEL at a time – Allosteric inhibition at other site • One side of cylinder is actively folding protein at a time
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1. GroEL/ATP complex at side A 2. Bind GroES on this side 7 ATP 7 ADP this side has a wider cavity but closed top other side has smaller cavity and open top 3. Side B ring binds unfolded protein GroES falls off of side A ADP falls off of side A 4. Side B ring binds 7 ATPs 5. GroES binds GroEL/ATP 7 ATP 7 ADP protein folding occurs 6. Side A ring binds 7 ATPs protein folding occurs 7 ATP 7 ADP (side A) 7 ADP & GroES (side B) falls off 7. Side A ring binds next unfolded protein
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• Switch side of ATP binding each time • Switch side of GroES binding for each folding rxn • Switch side of protein docking for each folding rxn Fink, Chaperone Mediated Folding, Physiological Reviews, 1999 Mechanism of Chaperonin Function
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Existence of Folding intermediates detected by NMR • Obtained by analysis of the disulfide bonding pattern of intermediates trapped during reoxidation of a 59 a.a. protein (bovine pancreatic trypsin inhibitor) • Barnase folding pathway-Fig. 6.4 & Kinemage • Role: transient structures in nascent chains – could initiate early steps in folding (funnels) • Biotechnology – problematic inclusion bodies
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Protein Stability (thermal) Protein engineering (mutagenesis) 1. S-S bridges a. -CH2-S-S-CH2- b. Analysis of all possibilities (many) c. Energy minimization to reduce to a few plausible candidates d. Site-selective mutations e. Protein synthesis f. Assay: example – T4 lysozyme (x-ray structure known) Reducing degrees of freedom (entropy) increases protein stability
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Protein Stability Cont… 2. Gly and Pro -Gly freedom -Pro Constraints (side chain is fixed by covalent bond to main chain -G ly Pro has propensity to increase stability (more delicate) Ala usually increase -Pro Ala usually decrease
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Protein Stability Cont… 3. Dipolar stability N-end (-a.a.) C-end (+a.a.) increase stability by mutating residues at N-end of helices from polar to negative (e.g. ASN ASP, SER ASP) Helix:
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Protein Stability 4. Hydrophobicity in the core (cavity) -Barnase (bacterial RNAse-110 a.a.) -structure by both x-ray and NMR
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Each side can accommodate one unfolded protein 1 GroES...

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