1. Folding Experiments2 UV absorbance of aromatic amino acids

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

3. A folded protein is easily recognized Protein domain

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

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

6. OXIDIZED Protein Disulfide Isomerase (PDI) 1)Forms protein’s initial S-S bonds in similar way (protein –SH attacks PDI S-S bond to give mixed disulfide) 2)Protein SH attacks protein-PDI mixed S-S bond to give protein S-S bond 3)Continues until protein in native S-S configuration and PDI cannot bind to exposed hydrophobic patches on the protein Folding Accessory Proteins6

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

8. Folding Accessory Proteins14 1.One ring binds ATP 7, 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 ATP 7 and substrate (must wait for cis ring to hydrolyse all 7) 4. ATP, substrate binding induces release of ES, ADP 7, and substrate 1 (presumably better folded)