Interaction of GroEL with conformational states of horse cytochrome c.
GroEL interacts with proteins in denatured states and promotes their efficient folding. To understand the conformational features required for the substrate, we studied the interactions of GroEL with various derivatives of horse cytochrome c including porphyrin-cytochrome c, apo-cytochrome c, and the three fragments containing the heme group, i.e. fragments 1-65, 1-38 and 11-21. Size-exclusion chromatography was performed, taking advantage of the heme absorption of the fluorescence label. Under low-salt conditions, significant binding to GroEL was observed for porphyrin-cytochrome c, apo-cytochrome c, and fragments 1-65 and 1-38, but not for intact cytochrome c or fragment 11-21. On the other hand, under conditions of high ionic strength, only apo-cytochrome c remained tightly bound. Whereas porphyrin-cytochrome c assumes a molten-globule-like compact conformation with a native-like far-UV CD, apo-cytochrome c shows a compact conformation without an ordered secondary structure. The far-UV CD spectra of the three fragments indicated that the fragments lack an ordered secondary structure. These results suggest that the fluctuating and exposed hydrophobic clusters of the substrates are responsible for the interaction, and that the interaction is modulated by electrostatic interaction. It is notable that these characteristics are similar to those of the interaction of cytochrome c derivatives with negatively charged phospholipid membranes, suggesting a common mechanism. Using fluorescence-labeled apo-cytochrome c, we also measured the kinetics of the interaction and estimated the association rate constant to be 5 x 10(7) M-1s-1. This relatively fast association rate constant indicates that the refolding rate of substrate protein is another important factor determining the interaction.
Department of Biology, Faculty of Science, Osaka University, Japan.
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M
Hoshino
Y
Y
Kawata
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Y
Goto
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D007526:Isoelectric Point; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D018834:Chaperonin 60; D003574:Cytochrome c Group; D000255:Adenosine Triphosphate