Abstract
The pH and ionic strength dependence of conformation of the COOH‐terminal fragment 206–316 (fragment FII) of thermolysin was monitored by far‐uv CD and difference absorption measurements. This fragment was shown previously to possess the properties of a protein domain, i.e., able to refold into a stable nativelike structure [Fontana, A., Vita, C. & Chaiken, I. M. (1983) Biopolymers 22, 69–78]. Analysis of the CD spectra in the pH range of 1–12 indicated that near pH 1, the conformation of fragment FII appears to be in an intermediate state (H) between the fully unfolded one (U) [the guanidine hydrochloride (Gdn · HCl)‐induced unfolded state] and the nativelike state (N—that attained at neutral pH). Quantitative analysis of secondary structure from CD spectra revealed that state H at 4°C is characterized by some 30% α‐helical structure, compared to 47% for state N. The heat‐ and Gdn · HCl‐mediated unfolding transitions of state H were fully reversible and characterized by little cooperativity, which is taken as an indication that state H corresponds to several species possessing different, and low, conformational stabilities. The midpoint transition from state H to N occurs near pH 2.5, implying that the acid transition results from the titration of carboxyl groups of the fragment with anomalously low p__K__, as would be expected for groups involved in specific salt bridges. Fragment FII at pH 1 (state H) may be induced to exhibit nearly the same degree of helicity of state N simply by increasing the ionic strength of the solution, thus reducing the repulsive interactions between positive charges within the highly charged fragment at pH 1. The results obtained emphasize the role of electrostatic interactions in the folding and stability of fragment FII and suggest a mechanism of folding of the fragment from U to N involving an intermediate state characterized by an assembly of fluctuating α‐helices.