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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
Stopped-flow device: ms resolution of early folding events
Monitor UV/Vis, fluorescence, or CD signals
Folding Accessory Proteins7
GRASP image of PDI (electrostatic surface potential: red=O- and blue=N+)
Folding Accessory Proteins6
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 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
Folding Accessory Proteins14
1. 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)