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Folding Experiments2

Folding Experiments2. UV absorbance of aromatic amino acids. Folding Experiments3. Circular Dichroism De is the difference in absorbance between left- and right-circularly polarized light…. Protein domain. A folded protein is easily recognized. Folding Experiments1.

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Folding Experiments2

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  1. Folding Experiments2 UV absorbance of aromatic amino acids

  2. Folding Experiments3 Circular Dichroism De is the difference in absorbance between left- and right-circularly polarized light…

  3. Protein domain A folded protein is easily recognized

  4. Folding Experiments1 Stopped-flow device: ms resolution of early folding events Monitor UV/Vis, fluorescence, or CD signals

  5. Folding Accessory Proteins7 GRASP image of PDI (electrostatic surface potential: red=O- and blue=N+)

  6. Folding Accessory Proteins6 • OXIDIZED Protein Disulfide Isomerase (PDI) • Forms protein’s initial S-S bonds in similar way (protein –SH attacks PDI S-S bond to give mixed disulfide) • Protein SH attacks protein-PDI mixed S-S bond to give protein S-S bond • Continues until protein in native S-S configuration and PDI cannot bind to exposed hydrophobic patches on the protein

  7. Folding Accessory Proteins13 ATP hydrolysis doubles volume of cis cavity, all 7 ATP hydrolysis at one time, mechanically linked subunits expand simultaneously, can accommodate 70kDa polypeptide chain Big cavity Small cavity

  8. Folding Accessory Proteins14 • One ring binds ATP7, substrate • GroES associates to cap it off • GroES binding causes hydrophobic • patches of cis ring to move to • interior GroEL position, depriving • substrate its binding sites • 2. It takes 13s for GroEL to • hydrolyse all 7 ATP and this • weakens affinity btw EL and ES • 3. Trans ring binds ATP7 and substrate • (must wait for cis ring to hydrolyse all 7) • 4. ATP, substrate binding induces • release of ES, ADP7, and substrate1 • (presumably better folded)

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