Conformational diversity of acid‐denatured cytochrome c studied by a matrix analysis of far‐UV CD spectra

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Abstract
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.