The contribution of electrostatic factors to the stabilization of the conformation of cytochrome c. Studies on the maleylated protein.

A Schejter,M Zuckerman,I Aviram

doi:10.1016/s0021-9258(18)50281-7

Abstract

All the lysines of horse heart cytochrome c were maleylated yielding a low spin product. At room temperature and low salt concentration, this product lacked the 695 nm absorption band and showed tryptophan fluorescence and circular dichroic spectra typical of denatured cytochrome c. The 695 nm band and the native tryptophan fluorescence and circular dichroic spectra were restored by addition of salts, their effectiveness being dependent on the charge of the cation. On low salt concentration, the 695 nm band was also restored by lowering the temperature. Studies of the temperature dependence of the 695 nm band indicate that the thermal denaturation of maleylated cytochrome c occurs at temperatures 60-70 degrees C lower than in the native protein. This implies a destabilization of the native conformation by 5.6 kcal/mol; a similar value is evidenced by comparative urea denaturation studies on the native and modified proteins. The results confirm the assumption that the native conformation of cytochrome c is mostly determined by interactions involving internal residues.

Highlights

All the lysines of horse heart cytochrome c were maleylated yielding a low spin product
The 695 nm band and the native tryptophan fluorescence and circular dichroic spectra were restored by addition of salts, their effectiveness being dependent on the charge of the cation
In the present study we report the effects of full maleylation of the lysines of horse cytochrome c on the conformation of the protein, and show that these effects can be reversed by proper manipulation of the temperature and electrolyte composition of the medium

Summary

Introduction

All the lysines of horse heart cytochrome c were maleylated yielding a low spin product. The electrostatic interactions take place between side chains distributed over the protein surface In principle, it is possible, to introduce perturbations into the electrostatic contribution to the conformation of the protein by suitable modification of the charged residues, without affecting the forces that operate in the interior domain of the molecule. It is possible, to introduce perturbations into the electrostatic contribution to the conformation of the protein by suitable modification of the charged residues, without affecting the forces that operate in the interior domain of the molecule This can be achieved, for example, by modifying the e-amino residues of horse cytochrome c.

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