Combined kinetic and thermodynamic analysis of α-helical membrane protein unfolding

The analytical toolkit developed for investigations into water-soluble protein folding has yet to be applied in earnest to membrane proteins. A major problem is the difficulty in collecting kinetic data, which are crucial to understanding any reaction. Here, we combine kinetic and thermodynamic studies of the reversible unfolding of an a-helical membrane protein to provide a definitive value for the reaction free energy and a means to probe the transition state. Our analyses show that the major unfolding step in the SDS-induced denaturation of bacteriorhodopsin involves a reduction in a-helical structure and proceeds with a large free-energy change; both our equilibrium and kinetic measurements predict that the free energy of unfolding in the absence of denaturant is +20 kcal-mol-1, with an associated m-value of 25 kcal-mol-1. The rate of unfolding in the absence of denaturant, kuH20, is surprisingly very slow (approximate to 10(-15) s(-1)). The kinetics also give information on the transition state for this major unfolding step, with a value for beta (m(f)/[m(f) + m(u)]) of approximate to 0.1, indicating that the transition state is close to the unfolded state. We thus present a basis for mapping the structural and energetic properties of membrane protein folding by mutagenesis and classical kinetics.