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

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
DOI: 10.1073/pnas.0705067104 Publication Date: 2007-11-20T02:15:10Z
ABSTRACT
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.
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