Conformational diversity of acid-denatured cytochrome c studied by a matrix analysis of far-UV CD spectra.
The singular value decomposition (SVD) analysis was applied to a large set of far-ultraviolet circular dichroism (far-UV CD) spectra (100-400 spectra) of horse heart cytochrome c (cyt c). The spectra were collected at pH 1.7-5.0 in (NH4)2SO4, sorbitol and 2,2,2-trifluoroethanol (TFE) solutions. The present purpose is to develop a rigorous matrix method applied to far-UV CD spectra to resolve in details conformational properties of proteins in the non-native (or denatured) regions. The analysis established that three basis spectral components are contained in a data set of difference spectra (referred to the spectrum of the native state) used here. By a further matrix transformation, any observed spectrum could be decomposed into fractions of the native (N), the molten-globule (MG), the highly denatured (D), and the alcohol-induced helical (H) spectral forms. This method could determine fractional transition curves of each conformer as a function of solution conditions, which gave the results consistent with denaturation curves of cyt c monitored by other spectroscopic methods. The results in sorbitol solutions, for example, suggested that the preferential hydration effect of the co-solvent stabilizes the MG conformer of cyt c. This report has found that the systematic SVD analysis of the far-UV CD spectra is a powerful tool for the conformational analysis of the non-native species of a protein when it is suitably supplemented with other experimental methods.
Konno
T
T
National Institute for Physiological Science, Myodaiji, Okazaki, Japan. konno@nips.ac.jp
D000143:Acids / Q000494:pharmacology; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects*; D017433:Protein Structure, Secondary; D013012:Sorbitol / Q000494:pharmacology; D014270:Trifluoroethanol / Q000494:pharmacology
D000143:Acids; D003574:Cytochrome c Group; D013012:Sorbitol; D014270:Trifluoroethanol; D000645:Ammonium Sulfate