EQUILIBRIUM UNFOLDING STUDIES OF BARSTAR - EVIDENCE FOR AN ALTERNATIVE CONFORMATION WHICH RESEMBLES A MOLTEN GLOBULE

The folding of the small protein barstar, which is the intracellular i nhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two confo rmations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectr oscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticit y measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule confo rmation. Fluorescence energy transfer experiments using 1-anilino-8-na phthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high conc entrations of denaturant are required to unfold the A form. For denatu ration by guanidine hydrochloride, the midpoint of the cooperative unf olding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2. 0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A f orm by guanidine hydrochloride or urea is complex and cannot be satisf actorily fit to a two-state (A half arrow right over half arrow left U ) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equil ibrium unfolding intermediate must be present on the unfolding pathway of A.