Structure and Dynamics of Molten Globular Intermediates Encountered during the Unfolding of Barstar

Abstract

The modelling of the folding and unfolding reactions of small proteins as two-state processes has provided us wealth of information on the kinetics and thermodynamics of these processes and the stability and pathway of folding. Hidden in this misleading simplicity are various intermediate states including the molten globular (MG) forms. The nature of solvation of MG forms has acquired considerable importance in the context of identifying the forces that drive protein folding and unfolding reactions.In this work we monitored the solvent accessibility during the unfolding of barstar, a small single domain protein, by following the bimolecular quenching constant (kq) associated with dynamic fluorescence quenching of the single and buried tryptophan (Trp-53) by either acrylamide or potassium iodide. A pico-second time-domain fluorimeter capable of acquiring data every 200 ms of unfolding was used. Analysis of the time-dependence of kq showed the presence of an intermediate with solvent accessibility very similar to that of the unfolded (U) state. However, structural indicators such as steady-state fluorescence intensity and time-resolved anisotropy of Trp-53 and near UV-CD indicated the presence of another intermediate within ∼ 10 ms, the zero time associated with the initiation of unfolding. Taken together, our data is consistent with the following model for the process of unfolding. N a DMG a WMG a U, where DMG and WMG refer to ‘dry’ and ‘wet’ MG forms, respectively. The demonstration of the presence of a DMG has strong implications in dissecting the overall process of folding and unfolding into individual steps, apart from revealing the forces that lead to protein folding.