Intermediate conformational states of apocytochrome c.
Horse apocytochrome c has been assumed to be a typical unfolded protein. At low ionic strength, the far- and near-UV circular dichroism spectra are typical of an unfolded protein at all pH values between 2 and 9. On the other hand, in the presence of high concentrations of salt, substantial secondary structure is present at both neutral and acidic pH. At low pH, perchlorate anion, either from salt or from acid, stabilizes an intermediate state (the A state) with secondary structure similar to that previously observed in the molten globule state of holocytochrome c. To further characterize the conformational states of apocytochrome c as a function of pH and salt, a fluorescence-labeled derivative was prepared, in which the two cysteine residues were labeled with N-(iodoacetyl)-N'-(5-sulfo-1- naphthyl)ethylenediamine(IAEDANS). The conformational transitions of the fluorescence-labeled apocytochrome c measured by circular dichroism were similar to those of unmodified apocytochrome c, indicating that the effects of the modification on the conformation and stability are small. Fluorescence energy transfer from tryptophan to the fluorescence label revealed several salt- and pH-dependent transitions. At very low ionic strength, apocytochrome c became compact as the pH was increased with a transition midpoint at pH 4.5. At acidic pH, increasing concentration of perchlorate induced a more compact state with a transition midpoint similar to that observed by circular dichroism. In contrast, at neutral pH, increasing perchlorate concentration had little effect on the compactness, as determined by a lack of change in the energy-transfer efficiency (but did increase the amount of secondary structure).(ABSTRACT TRUNCATED AT 250 WORDS)
Hamada
D
D
Department of Biology, Faculty of Science, Osaka University, Japan.
Hoshino
M
M
Kataoka
M
M
Fink
A L
AL
Goto
Y
Y
D001059:Apoproteins / Q000737:chemistry*; D002942:Circular Dichroism; D003545:Cysteine; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006851:Hydrochloric Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009282:Naphthalenesulfonates; D009994:Osmolar Concentration; D010472:Perchlorates / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014965:X-Rays
D001059:Apoproteins; D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010472:Perchlorates; C006421:1,5-I-AEDANS; D045304:Cytochromes c; D003545:Cysteine; D006851:Hydrochloric Acid