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

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)2S04r 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.

Structural plasticity of 4-α-helical bundles exemplified by the puzzle-like molecular assembly of the Rop protein

“…Melting curves were obtained from the CD signal at 222 nm for temperatures 10-90°C with a temperature increase of 80°C/h and a waiting time of 2 s for stabilization. SVD analysis of the far-UV CD spectra was performed with the program SVD1 (44). More details are provided are provided in SI Materials and Methods.…”

Application of multivariate resolution methods to the study of biochemical and biophysical processes

“…(1) [14,[35][36][37]. MCR allows the simultaneous analysis of several data matrices obtained from one or more experiments simultaneously monitored with several spectroscopies (for molecular absorption, CD, Xuorescence, etc.).…”

Fluorinated alcohol, the third group of cosolvents that stabilize the molten‐globule state relative to a highly denatured state of cytochrome c

“…Recent research has emphasized the biological importance of nonnative conformations of protein ranging from various denatured conformations to aggregated forms~Bychkova et al., 1988;Sipe, 1992;Beermann et al, 1998;Prusiner et al, 1998;Sigler et al, 1998;Dobson, 1999;Ellis, 1999!. Nonnative states are easily achieved by solvent perturbation~Tanford, 1968;Timasheff, 1993;Konno, 1998!. An extensively studied cosolvent that modifies the protein conformation is alcohol.…”

“…Studies on alcohol effects provide insights into biologically important events because the alcohol solution mimics the environment of biomembrane~Bychkova et al, 1996!, modifies folding pathways of proteins~Lu et al, 1997;Chiti et al, 1999! and induces the assembly of biologically-relevant peptides Mchaourab et al, 1993;Cort et al, 1994!. Spatial dimensions of the highly helical alcohol-denatured state of protein have recently been studied extensively~Kamatari et , 1998Hoshino et al, 1997;Gast et al, 1999!, reveal-ing that the helical denatured state at high concentrations of alcohol is expanded to an extent comparable to that of the denaturantinduced highly denatured state. When alcohol was added to the solution of a highly unstructured denatured protein, it did not induce compact conformation~Kamatari et al, 1996conformation~Kamatari et al, , 1999!.…”

“…Horse heart cyt c consists of 104 amino acid residues and a heme ring, whose phase properties were well studied as a function of pH and solvent compositions~Ohgushi & Wada, 1983;Goto et al, 1990;Jeng & Englander, 1991;Kamatari et al, 1996;Konno, 1998!. We monitored conformational changes of acid-denatured cyt c at pH 1.9 using far-ultraviolet~UV!…”