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

Abstract

The analytical toolkit developed for investigations into watersoluble 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 ␣-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 ␣-helical structure and proceeds with a large freeenergy change; both our equilibrium and kinetic measurements predict that the free energy of unfolding in the absence of denaturant is ؉20 kcal⅐mol ؊1 , with an associated m-value of 25 kcal⅐mol ؊1 . The rate of unfolding in the absence of denaturant, ku H2O , is surprisingly very slow (Ϸ10 ؊15 s ؊1 ). The kinetics also give information on the transition state for this major unfolding step, with a value for ␤ (mf/[mf ؉ mu]) of Ϸ0.1, 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.bacteriorhodopsin ͉ folding ͉ free energy ͉ transition state T he folding and assembly of integral membrane proteins is a fundamental process within biological systems, yet surprisingly few quantitative studies of membrane protein folding have been performed. Many transmembrane ␣-helical proteins are unstable once they are displaced from the biological membrane, and our limited understanding of the factors affecting folding and stability presents a major barrier to studies of structure and function.Bacteriorhodopsin (bR), a light-driven proton pump from the purple membrane (PM) of Halobacterium salinarum, is the current paradigm for studies of ␣-helical membrane protein folding (1, 2). The native purple chromophore is formed by the covalent attachment of the cofactor retinal at K216 and offers a direct, quantitative measure of the folded state of the protein because it is strongly influenced by retinal isomerization and noncovalent binding pocket interactions (i.e., refs. 3 and 4).The unfolding and refolding of bR in lipid/detergent mixtures was first reported in the seminal work of London and Khorana (5). bR was found to unfold in SDS from the native purple state by way of an intermediate to give denatured apoprotein, bacterioopsin (bO), and bR was efficiently reconstituted from bO by replacing SDS with renaturing micelles. The kinetics of refolding have been investigated in considerable detail (1, 6, 7). A recent equilibrium study of this denaturation of bR (8) has also demonstrated that the free-energy change of unfolding could be measured. A full kinetic and thermodynamic study of unfolding and folding is, however, vital for a complete understanding of the reaction mechanism, but it has yet to be achieved. Here, we present such a study and prob...

Measuring membrane protein stability under native conditions

“…bR contains a covalently bound retinal chromophore that complicates unfolding analysis because it can slowly hydrolyze off in the SDS unfolded protein (12). We and others originally measured an apparent equilibrium between the folded bR (bR F ) and the unfolded protein with the intact chromophore (bR U ) (7,13). We recently determined, however, that the rate of refolding in the transition zone was too slow compared with the retinal hydrolysis rate to obtain a true equilibrium (10).…”

“…The Booth laboratory has pioneered and extensively studied the refolding kinetics of bR from an SDS-denatured state (6)(7)(8). We introduced SDS unfolding to measure the thermodynamic stability of the membrane enzyme diacylglycerol kinase (9) and a similar approach can be used to measure bR thermodynamic stability (10,11).…”

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…SDS-solubilized BR often serves as a starting point for folding studies [54]. A comprehensive characterization of this state is therefore crucial for deciphering the mechanism by which BR refolds and inserts into the lipid bilayer.…”

Stable folding core in the folding transition state of an α-helical integral membrane protein

“…bR can be reversibly unfolded according to a two-state reaction in mixed lipid/detergent micelles (7)(8)(9) analogous to the unfolding of aqueous proteins in urea or guanidinium. We recently used this folding system to study a series of Ala mutations through transmembrane helix B (9) and carried out a classical Φ-value analysis in order to obtain information on the nature of the folding transition state (10).…”