Folding Experiments2UV absorbance of aromaticamino acids

Folding Experiments3

Circular Dichroism is the difference in absorbancebetween left- and right-circularlypolarized light…

A folded protein is easily recognized

Protein domain

Folding Experiments1

Stopped-flow device: ms resolution of early folding events

Monitor UV/Vis, fluorescence, or CD signals

GRASP image of PDI (electrostatic surface potential: red=O- and blue=N+)

Folding Accessory Proteins7

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 bond3) Continues until protein in native S-S configuration and PDI cannot bind to

exposed hydrophobic patches on the protein

Folding Accessory Proteins6

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

Folding Accessory Proteins141. One ring binds ATP7, substrate GroES associates to cap it off

GroES binding causes hydrophobicpatches 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 thisweakens 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)