Independent folding of the carboxyl-terminal fragment 228-316 of thermolysin.
The COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
D001407:Bacillus / Q000201:enzymology*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D005779:Immunodiffusion; D007163:Immunosorbent Techniques; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D013820:Thermolysin
D010446:Peptide Fragments; D013820:Thermolysin