Combined kinetic and thermodynamic analysis of alpha-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 alpha-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 alpha-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 ( approximately 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 approximately 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.
Curnow
Paul
P
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. p.curnow@bristol.ac.uk
Booth
Paula J
PJ
D001436:Bacteriorhodopsins / Q000737:chemistry; D002793:Cholic Acids; D002942:Circular Dichroism; D003902:Detergents; D004134:Dimyristoylphosphatidylcholine; D007700:Kinetics; D008823:Micelles; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate; D013816:Thermodynamics
D002793:Cholic Acids; D003902:Detergents; D008823:Micelles; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D004134:Dimyristoylphosphatidylcholine