Expression of functionaly of α-chymotrypsin. Effects of guanidine hydrochloride and urea in the onset of denaturation

Abstract

Crystals of alpha-chymotrypsin (CHT) at equilibrium in solutions of 2.0 M guanidine hydrochloride and 3.0 M urea at pH 3.6 were prepared, three-dimensional X-ray intensities were measured, and difference electron-density maps were calculated and examined. The guanidine hydrochloride derivative displayed changes occurring exclusively on the surface of the protein. The difference peaks represented mostly small changes in various protein surface groups and in the adjacent solvent regions, and some displayed convincing evidence of binding of the guanidinium ion to the protein. The urea difference map likewise showed that changes had occurred on the surface of the protein, but also that numerous changes in the structure occurred in the hydrophobic interior of the CHT molecule. Further, the urea difference map contained evidence for two kinds of interactions of urea with protein groups. There are examples of bound urea either causing or accompanying structural changes and examples of urea binding with no accompanying changes to the protein. Examples of both kinds of binding were observed in both the surface regions and in the hydrophobic interior of the molecule. From an examination of these two derivatives, it is clear that guanidine hydrochloride and urea unfold proteins by different mechanisms.

A structural basis for the interaction of urea with lysozyme

“…Here, urea interacts with the enzyme via multiple hydrogen bonds made primarily to peptide groups and polar side chains. In contrast to results from a similar crystallographic study of denaturant binding to achymotrypsin (Hibbard & Tulinsky, 1978), none of the urea binding sites on HEWL are particularly hydrophobic in nature. Further detailed experimental studies on proteins less resistant to denaturation are needed to get a better insight into the molecular mechanisms of urea's action.…”

How Do Volatile Anesthetics Inhibit Ca2+-ATPases?

“…The findings suggest selectivity in the interactions between anesthetics and proteins and a requirement for compatibility between the nonpolar sites available in the protein (proper steric arrangement of amino acid side chains) and the intruding molecule (size, shape, and polarizability). The Ca 2ϩ -ATPase seems to have a fitting nonpolar site(s) available to all five volatile anesthetics studied in our laboratory (present paper and in Kosk-Kosicka and Roszczynska, 1993) and to xenon (Franks et al, 1995) -ATPase molecule their binding is expected to affect internal motions and conformational substates of the enzyme (Schoenborn, 1965;Brown et al, 1976;Hibbard and Tulinsky, 1978;Tilton et al, 1984;Lim et al, 1994). The observed effects on total tryptophan fluorescence might then reflect the invasion that results in subtle conformational changes including tryptophan environment.…”

“…To accommodate the intruding anesthetic the protein molecule undergoes structural rearrangement that results in the loss of its Ca 2ϩ -ATPase activity. In proposing this mechanism of anesthetic action on the Ca 2ϩ -ATPase we are considering multiple x-ray diffraction and NMR data that have demonstrated binding of gaseous anesthetics and other molecules, including urea and steroids, in hydrophobic "cavities" in the interior of several proteins (Schoenborn, 1965;Schoenborn et al, 1965;Schoenborn and Featherstone, 1967;Nunes and Schoenborn, 1973;Brown et al, 1976;Sachsenheimer et al, 1977;Hibbard and Tulinsky, 1978;Tilton and Kuntz, 1982;Tilton et al, 1984;Otting et al, 1991;Arevalo et al, 1994;Jain et al, 1994;Williams et al, 1994;Hubbard et al, 1994).…”

Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride

“…The change in heat capacity of unfolding measured with heat unfolding and denaturant-induced unfolding are different, indicating that chemical denaturant may participate directly in the process (41). Discrete denaturant binding sites and low B-factors of surface residues have also been observed in the X-ray crystal structures of proteins in the presence of GdmCl (7,8,42).…”