Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure.
The COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002918:Chymotrypsin; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D011487:Protein Conformation; D013381:Subtilisins; D013696:Temperature; D013820:Thermolysin / Q000276:immunology; D014357:Trypsin
D000596:Amino Acids; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; D013820:Thermolysin