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The Transition State for Integral Membrane Protein Folding

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The Transition State for Integral Membrane Protein Folding The transition state for integral membrane protein folding Paul Curnow1 and Paula J. Booth1 Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom Edited by Alan Fersht, University of Cambridge, Cambridge, United Kingdom, and approved November 21, 2008 (received for review July 18, 2008) Biology relies on the precise self-assembly of its molecular com- water-soluble proteins. In particular, there are few quantitative ponents. Generic principles of protein folding have emerged from studies of the kinetics and thermodynamics of membrane protein extensive studies on small, water-soluble proteins, but it is unclear folding. Yet, understanding these folding parameters gives valu- how these ideas are translated into more complex situations. In able information on a major problem: how to fold and stabilize particular, the one-third of cellular proteins that reside in biological membrane proteins for structural and functional studies. membranes will not fold like water-soluble proteins because mem- A ⌽ value is a measure of the change in activation energy, brane proteins need to expose, not hide, their hydrophobic sur- relative to the change in the overall free energy of folding, faces. Here, we apply the powerful protein engineering method of induced by the directed mutation of a single amino acid. ⌽ values ⌽-value analysis to investigate the folding transition state of the are expected to fall between one and zero (these extreme values alpha-helical membrane protein, bacteriorhodopsin, from a par- mean the change in the free energy of the transition state is the tially unfolded state. Our results imply that much of helix B of the same as either that in the folded or unfolded state, respectively). seven-transmembrane helical protein is structured in the transition Based on this energetic perturbation, the magnitude of ⌽ can be state with single-point alanine mutations in helix B giving ⌽ values interpreted as the extent of native interactions at the site of the >0.8. However, residues Y43 and T46 give lower ⌽ values of 0.3 mutated residue in the transition state, with a value of one and 0.5, respectively, suggesting a possible reduction in native indicating native energetics and interactions. Intermediate ⌽ structure in this region of the helix. Destabilizing mutations also values are often observed (6), suggesting that a proportion of increase the activation energy of folding, which is accompanied by native-like contacts are formed or that parallel reaction path- BIOPHYSICS an apparent movement of the transition state toward the partially ways exist. unfolded state. This apparent transition state movement is most We present a ⌽-value analysis of the major folding step of an likely due to destabilization of the structured, unfolded state. ␣-helical membrane protein. The protein of choice for this study These results contrast with the Hammond effect seen for several is bacteriorhodopsin (bR), a light-activated proton pump from water-soluble proteins in which destabilizing mutations cause the the purple membrane of Halobacterium salinarum. We have transition state to move toward, and become closer in energy to, previously established conditions for reversible folding of bR in the folded state. We thus introduce a classic folding analysis mixed lipid/detergent (1,2-dimyristoyl-sn-glycero-3-phospho- method to membrane proteins, providing critical insight into the choline, DMPC)/3-[(3-cholamidopropyl)dimethylammonio]-1- folding transition state. propanesulfonate, CHAPS) micelles (10). Currently, bR is the only helical membrane protein that meets the criteria for ⌽-value phi value ͉ kinetics ͉ thermodynamics ͉ protein engineering analysis, namely (i) it can be partially unfolded with a reduction in secondary structure to the apoprotein bacterioopsin (bO) by sodium dodecylsulfate (SDS) in a microscopically reversible, rotein folding plays a central role in biology. Folding inves- cooperative, two-state reaction (10, 11); (ii) this process can be tigations provide key information on protein structure and P described by linear free-energy relationships to obtain the dynamics, while protein misfolding can have serious disease H O overall free energy change of unfolding (⌬Gu 2 ) and the folding implications (1, 2). To fold correctly, proteins must overcome an H O and unfolding rate constants in the absence of denaturant (kf 2 activation barrier to pass through a high-energy transition state. H O and k 2 ); and (iii) the effect of single point mutations can be Understanding the nature of this folding transition state is u interpreted in the context of a detailed native-state structure (12). important in resolving how a protein folds to a stable and bR is a relatively large protein (248 aa) with seven transmem- functional native structure (3, 4). brane ␣ helices, so we begin our survey of the folding transition The most powerful method available to probe the structure state by determining the ⌽ values of a discrete region of the and energetics of the folding transition state combines site- protein. We use an alanine scan to investigate a single helix of directed mutagenesis, equilibrium thermodynamics, and kinetic ⌽ bR, helix B, which is thought to form early during folding and dataina -value analysis. This approach has revolutionized does not make extensive interactions with the retinal cofactor. protein folding studies by providing a quantitative description of the environment experienced by individual side chains in the Results folding transition state and has been applied to the folding of Kinetic and Thermodynamic Measurements. The absorbance band of many small, water-soluble proteins (3, 5–8). However, the ⌽ the retinal chromophore of bR reports on the conformational -value method has yet to be applied to larger integral mem- state of the protein. bR is purple when folded with an absorbance brane proteins. Integral membrane proteins are a special case in protein folding because they are adapted to the lipid bilayer rather than Author contributions: P.C. and P.J.B. designed research; P.C. performed research; P.C. to the cytoplasmic milieu (9). The sequences of transmembrane analyzed data; and P.C. and P.J.B. wrote the paper. regions are biased in favor of hydrophobic amino acids to match The authors declare no conflict of interest. the low dielectric of the membrane interior. This presents This article is a PNAS Direct Submission. experimental difficulties for in vitro studies of protein folding as 1To whom correspondence may be addressed. E-mail: [email protected] or the proteins need to be solubilized in lipids or detergents and are [email protected]. often resistant to common denaturants such as urea. Conse- This article contains supporting information online at www.pnas.org/cgi/content/full/ quently, the current understanding of membrane protein folding 0806953106/DCSupplemental. is poor compared with the advanced state of knowledge for © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806953106 PNAS ͉ January 20, 2009 ͉ vol. 106 ͉ no. 3 ͉ 773–778 Downloaded by guest on October 4, 2021 band at 560 nm. This 560-nm absorbance decays when bR in DMPC/CHAPS is unfolded by the addition of SDS. We have previously shown that these absorption changes correlate with a reduction in secondary structure (10). The reaction we study here is the transition between the partially denatured apoprotein state in SDS (referred to as the SDS-unfolded state) and the folded bR state. We have previously shown that this process corresponds to the final, major folding step of bR (10) and behaves as a two-state reaction. The SDS state is not completely unfolded, but has helical content equivalent to approximately four of the native seven helices of folded bR (13). Thus, the SDS-unfolded state has much more ordered structure than the urea- or guanidinium chloride-induced unfolded states fre- quently used in water-soluble folding studies. The folding reac- tion we study here is more akin to the later stages of multistate water-soluble protein folding, that is, from a structured inter- mediate to a folded state. The solvent for the bR unfolding reaction is also relatively complex; folded bR is solubilized in DMPC/CHAPS micelles and begins to unfold when SDS is added and forms mixed DMPC/CHAPS/SDS micelles. At higher SDS concentrations, SDS micelles dominate and solubilize the partly unfolded bR state. Free energies of unfolding of wild-type (WT) and mutant bR were obtained as described previously from equilibrium dena- turation curves (11) as well as from time-resolved, stopped-flow absorption measurements of the folding and unfolding rates (10). Both the equilibrium and kinetic data fit a two-state reaction, and linear free energy relationships were observed, enabling extrapolation to zero SDS denaturant. Linear Free Energy Relationships for Alanine Mutants. ⌽-value anal- ysis is most successful when single point mutations are intro- H O duced that perturb the overall free energy of unfolding, ⌬Gu 2 , by Ͼ0.6 kcal.molϪ1 (14). On this basis, we selected nine of 24 residues within bR helix B that are potentially suitable for ⌽-value analysis when mutated to alanine (Ϫ0.6 kcal.molϪ1 Յ H O Ϫ1 ⌬⌬Gu 2 Յ Ϫ1.6 kcal.mol ) (11). These are D36A, K41A, F42A, Y43A, T46A, T47A, I52A, F54A, and M60A; several of these would be considered relatively extreme mutations in water soluble proteins but reflect the prevalence of large aromatic side chains in integral membrane proteins. The logarithms of the folding and unfolding rate constants (kf and ku, respectively) of each mutant were linear with SDS (10), giving a characteristic chevron plot (see Fig. 1A and Table 1). Because the experimen- tally observed rate constant kobs is the sum of kf and ku, the characteristic ‘‘downward arrow’’ of the chevron plot can be fit by the sum of two linear functions and extrapolation of these lines to the y axis gives the folding and unfolding rates in the H O H O absence of denaturant, termed kf 2 and ku 2 , respectively.
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