TITRATION PROPERTIES AND THERMODYNAMICS OF THE TRANSITION-STATE FOR FOLDING - COMPARISON OF 2-STATE AND MULTISTATE FOLDING PATHWAYS

CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have ex amined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In add ition to the standard thermodynamic quantities, we have measured the o verall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were abl e to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium un folding: the enthalpy of activation of unfolding at different values o f acid pH were plotted against the average temperature of each determi nation. Our results show that the transition state of CI2 has lost mos t of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water mo lecules from entering some parts of the hydrophobic core. The properti es of the transition state of CI2 are then compared with the major fol ding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured in termediate (D-phys or I). CI2 folds from a largely unstructured denatu red state under physiological conditions via a transition state which is compact but relatively uniformly unstructured, with tertiary and se condary structure being formed in parallel. We term this an expanded p athway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transit ion state that has islands of folded elements of structure. We term th is a compact pathway. These two pathways may correspond to the two ext reme ends of a continuous spectrum of protein folding mechanisms. Alth ough the properties of the two transition states are very different, t he activation barrier for folding (D-phys --> double dagger) is very s imilar for both proteins. (C) 1996 Academic Press Limited