STABILITY AND FOLDING PROPERTIES OF A MODEL BETA-SHEET PROTEIN, ESCHERICHIA-COLI CSPA

Although beta-sheets represent a sizable fraction of the secondary str ucture found in proteins, the forces guiding the formation of beta-she ets are still not well understood. Here we examine the folding of a sm all, all beta-sheet protein, the E. coli major cold shock protein CspA , using both equilibrium and kinetic methods. The equilibrium denatura tion of CspA is reversible and displays a single transition between fo lded and unfolded states. The kinetic traces of the unfolding and refo lding of CspA studied by stopped-flow fluorescence spectroscopy are mo noexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding: is largely ind ependent of urea concentration. This suggests that the transition stat e ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compa ct transition state and on an unusual structural feature of CspA, a so lvent-exposed cluster of aromatic side chains, we propose a novel fold ing mechanism for CspA. We have also investigated the possible complic ations that may arise from attaching polyhistidine affinity tags to th e carboxy and amino termini of CspA.