Compactness of thermally and chemically denatured ribonuclease A as revealed by volume and compressibility

Abstract

The conformational changes of ribonuclease A due to thermal and guanidine hydrochloride denaturation were monitored by means of precise density and sound velocity measurements. It was found that the apparent molar volume decreased but the adiabatic compressibility increased on thermal denaturation under acidic conditions (pHs 1.60, 1.90, and 2.08). On the other hand, guanidine hydrochloride denaturation (pH 2.00) brought about large decreases in the compressibility and apparent molar volume. These results indicate that the conformation of the denatured protein is greatly different between the two types of denaturation: the thermally denatured state corresponds to the structure with enhanced thermal fluctuation having a residual secondary structure and a high local concentration of nonpolar groups exposed, but the guanidine hydrochloride denaturation leads to exposure of a large amount of amino acid residues, resulting in an increase in hydration and a decrease in the internal cavity. The compressibility changes due to both types of denaturation were not correlated to a loss of the secondary structure, as judged by means of circular dichroism. These findings suggest that the compactness and thermal fluctuation of the protein cannot be described by a two-state denaturation model and that there are some molten-globule-like intermediates in the denaturation processes.

Chemically crosslinked protein dimers: Stability and denaturation effects

“…It is not entirely unexpected that the thermal and solvent denaturations are different. It is well known that the denatured states resulting from these two processes are different (Dill & Shortle, 1991;Shortle, 1993;Xie & Freire, 1994;Tamura & Gekko, 1995). For example, many researchers agree that the thermal-denatured state is generally more compact than the solvent-denatured state, preserving significant amounts of secondary structure.…”

Volumetric and Viscometric Behaviour of some Amino Acids and their Group Contributions in Aqueous Tetramethylammonium Bromide at Different Temperatures

“…The interactions occurring between proteins and salts are difficult to predict due to the complex protein structure. Amino acids which are the building blocks of protein are used as model compounds to study the effect of these electrolytes [2][3][4][5]. Therefore, study of thermodynamic properties of amino acids can provide valuable information regarding the conformational stability and interactions of proteins in solutions.…”

A large compressibility change of protein induced by a single amino acid substitution

“…A novel measure of the flexibility is compressibility because it is directly linked to the volume fluctuation (Cooper, 1976). Although this type of fluctuation is a thermodynamic one involving the effects of internal cavity and surface hydration, the partial specific adiabatic compressibility, a,, has been confirmed to sensitively reflect the characteristics or compactness of protein structure (Gekko & Noguchi, 1979;Gekko&Hasegawa, 1986,1989Sarvazyan, 1991;Tamuraetal., 1993;Tamura & Gekko, 1995). In the present study, the 6, values were measured for a series of single amino-acid substituted Reprint requests to: Kunihiko Gekko, Department of Materials Science, Faculty of Science, Hiroshima University, Higashi-Hiroshima 739, Japan; e-mail: gekko@alphaOl .sci.hiroshirna-u.ac.jp.…”

“…The compressibility measurements on DHFR and AspAT enzymes were performed without NADPH and with pyridoxal 5'-phosphate, at 15 and 25 "C, respectively. The apparatus and procedures used for compressibility measurements were same as our previous studies (Gekko & Noguchi, 1979;Gekko & Hasegawa, 1986, 1989Tamura et al, 1993;Tamura & Gekko, 1995). The sound velocity in protein solution, u, and the solution density, d, were measured by means of the "sing-around pulse method" at 5 MHz and with a precision density meter, DMA-02C (Anton Paar, Gratz), respectively.…”