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

• DOI: 10.1073/pnas.0705067104
• Publication Date: 2007-11-20T02:15:10Z
• Authors: Paul Curnow; Paula J. Booth
• Keywords: 0301 basic medicine; Protein Structure; Secondary; 570; Protein Denaturation; 0303 health sciences; Circular Dichroism; Detergents; Sodium Dodecyl Sulfate; Cholic Acids; Protein Structure, Secondary; Kinetics; 03 medical and health sciences; Bacteriorhodopsins; Thermodynamics; Dimyristoylphosphatidylcholine; Micelles

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 α-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 α-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, k u

H 2 O

, is surprisingly very slow (≈10 −15 s −1 ). The kinetics also give information on the transition state for this major unfolding step, with a value for β ( m f /[ m f + m u ]) of ≈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.