Beyond the EX1 limit: probing the structure of high-energy states in protein unfolding.

Hydrogen exchange kinetics in native solvent conditions have been used to explore the conformational fluctuations of an immunoglobulin domain (CD2.domain1). The global folding/unfolding kinetics of the protein are unaltered between pH 4.5 and pH 9.5, allowing us to use the pH-dependence of amide hydrogen/deuterium exchange to characterise conformational states with energies up to 7.2kcal/mol higher than the folded ground state. The study was intended to search for discreet unfolding intermediates in this region of the energy spectrum, their presence being revealed by the concerted exchange behaviour of subsets of amide groups that become accessible at a given free energy, i.e. the spectrum would contain discreet groupings. Protection factors for 58 amide groups were measured across the pH range and the hydrogen-exchange energy profile is described. More interestingly, exchange behaviour could be grouped into three categories; the first two unremarkable, the third unexpected. (1) In 33 cases, amide exchange was dominated by rapid fluctuation, i.e. the free energy difference between the ground state and the rapidly accessed open state is sufficiently low that the contribution from crossing the unfolding barrier is negligible. (2) In 18 cases exchange is dominated by the global folding transition barrier across the whole pH range measured. The relationship between hydroxyl ion concentration and observed exchange rate is hyperbolic, with the limiting rate being that for global unfolding; the so-called EX1 limit. For these, the free energy difference between the folded ground state and any rapidly-accessed open state is too great for the proton to be exchanged through such fluctuations, even at the highest pH employed in this study. (3) For the third group, comprising five cases, we observe a behaviour that has not been described. In this group, as in category 2, the rate of exchange reaches a plateau; the EX1 limit. However, as the intrinsic exchange rate (k(int)) is increased, this limit is breached and the rate begins to rise again. This unintuitive behaviour does not result from pH instability, rather it is a consequence of amide groups experiencing two processes; rapid fluctuation of structure and crossing the global barrier for unfolding. The boundary at which the EX1 limit is overcome is determined by the equilibrium distribution of the fluctuating open and closed states (K(O/C)) and the rate constant for unfolding (k(u)). This critical boundary is reached when k(int)K(O/C)=k(u). Given that, in a simple transition state formalism: k(u)=K(#)k' (where K(#) describes the equilibrium distribution between the transition and ground state and k' describes the rate of a barrierless rearrangement), it follows that if the pH is raised to a level where k(int)=k', then the entire free energy spectrum from ground state to transition state could be sampled.