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 reaction we study here is more akin to the later stages of multistate water-soluble protein folding, that is, from a structured intermediate 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 denaturation 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 analysis 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. These kinetic parameters were used to calculate the difference H O in the overall free-energy change upon mutation, ⌬⌬Gu 2 , and H O the values were very similar to ⌬⌬Gu 2 from equilibrium experiments, showing that the extrapolations of both kinetic and equilibrium data to zero denaturant are in good agreement. The H O consistency of the ⌬⌬Gu 2 values determined from kinetic and equilibrium data, also confirms that the mutants fold by a two-state process as previously established for wild type bR (see Fig. 1 B and Table 1). The refolding yield of all mutants was close to wild-type levels at Ͼ75% (see Supporting Information (SI) Fig. S1). Fig. 1. Kinetics of folding and unfolding. (A) Chevron plot of a typical Fig. 1C shows that destabilising mutations have equal and mutant (open circle and solid line) compared with WT bR (dashed line). (B) H O chev ⌬⌬Gu 2 derived from kinetic chevron plots (⌬⌬Gu ) and equilibrium mea- opposite effects on the observed folding and unfolding rates eqm H O H O surements (⌬⌬Gu ). The negative sign of ⌬⌬Gu indicates destabilisation and (kf 2 and ku 2 , respectively). As the folding rate slows, the a reduction in the magnitude of ⌬Gu. Correlation coefficient, 0.95; gradient, unfolding rate becomes faster to a similar degree, with gradients H O 0.9 Ϯ 0.1. (C) Linear changes in overall folding and unfolding rates, kf 2 (filled H O of 0.86 and 0.69, respectively. The mutations T47A and I52A circles) and ku 2 (open circles), respectively] for destabilizing mutants. For H O H O induced additional kinetic phases and unusually fast unfolding kf 2 , gradient, 0.86 Ϯ 0.3; correlation coefficient, 0.74; for ku 2 , gradient, rates that are omitted from the dataset. 0.69 Ϯ 0.3; correlation coefficient, 0.68.

774 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806953106 Curnow and Booth Downloaded by guest on October 4, 2021 0.02 0.01 0.01 0.01 0.01 0.01 0.02 0.03 ʈ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ␤ 0.1 0.007 0.3 0.02 0.07 0.003 0.1 0.06 0.1 0.002 0.4 0.01 0.18 0.04 0.401¶ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ F ⌽ 0.3 0.8 0.3 0.7 0.4 1 0.7 1 0.2 0.2 0.6 0.6 0.4 0.3 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ eqm§ F ⌽ 1 0.8 0.9 0.7 0.7 0.8 0.8 0.9 0.7 1 0.3 0.3 2.5 0.5 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ chev§ F ⌽ , Ϫ 1 0.28 0.9 0.47 0.9 0.56 0.8 0.40 1.0 0.21 1.3 0.95 0.4 2.4 0.5 eqm† Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ u ), respectively. ⌬⌬ G kcal ⅐ mol Ϫ 1.60 Ϫ 1.51 Ϫ 1.45 Ϫ 0.61 Ϫ 1.23 Ϫ 1.79 Ϫ 2.51 Oeqm 2 H , u Ϫ 1 1.5 1.0 1.2 0.41 0.45 1.0 13 chev† Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ u ⌬⌬ G kcal ⅐ mol Ϫ 1.42 Ϫ 1.11 Ϫ 1.48 Ϫ 0.57 Ϫ 0.96 Ϫ 1.37 Ϫ 2.58 , ‡ Ϫ 1 0.52 0.27 0.40 0.22 0.43 0.22 0.89 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ TS-U ⌬⌬ G BIOPHYSICS kcal ⅐ mol Ϫ 1.31 Ϫ 0.98 Ϫ 1.21 Ϫ 0.56 Ϫ 1.30 Ϫ 0.52 Ϫ 1.26 ) and equilibrium data ( ⌬ G , Ϫ 1 0.27 ------0.08 0.26 0.58 0.71 0.49 0.19 1.23 2.02 Ochev eqm 2 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ H u U-F m ⌬ G kcal ⅐ mol , Ϫ 1 0.2 28.30 0.19 25.61 0.42 25.56 0.53 25.46 0.35 27.94 0.08 28.37 0.93 25.85 2.40 20.97 . Oeqm† Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 2 SDS H

␹ u kcal ⅐ mol ⌬ G Ϫ 1 *, 0.38 20.57 0.85 18.97 0.59 19.06 0.63 19.12 0.56 19.96 0.33 19.34 0.57 18.78 3.40 18.06 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ chev U-F is given. m kcal ⅐ mol U-F , m

Ϫ 1 Fig. 2. Changes in denaturant response upon mutation. (A) Dependence of 10 24.83 20 23.10 17 23.19 14 21.95 14 23.82 9 23.37 13 23.46 86 21.27 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ kinetic m values, reﬂecting the dependence of the folding and unfolding rates Ochev† . 2

O ⌬ H O

H 2 2 on SDS (mTS-U and mTS-F, respectively) on the overall free energy ( Gu ). For u H u mTS-U, gradient, Ϫ0.83 Ϯ 0.4; correlation coefﬁcient, 0.66; for mTS-F, gradient, kcal ⅐ mol ⌬ G values were obtained at 0.431

⌬ G 0.70 Ϯ 0.3; correlation coefficient, 0.68. The Inset shows the trend for the ⌽ Ϯ Ϫ 1 overall m value, mU-F, where gradient, 1.54 0.3; correlation coefficient, 0.92. 1.34 ------0.30 19.73 0.31 ------0.77 18.31 0.54 18.62 0.54 18.25 0.44 19.16 0.29 18.77 0.40 18.36 3.31 17.15 *, Errors from chevron plot curve fitting. (B) Dependence of ␤ (mTS-U/mU-F)on Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ

TS-F ⌬⌬GTS-U. ⌬⌬GTS-U [i.e., ⌬GTS-U (WT) - ⌬GTS-U (MUT)] has a negative sign because m ⌬GTS-U (MUT) Ͼ⌬GTS-U (WT). Gradient, 0.06 Ϯ 0.004; correlation coefﬁcient, 0.98. kcal ⅐ mol 8 ; only the magnitude of . Very similar SDS

␹ 7 , and , 4.3E-16 22.73 1.2e-15 22.93 1.0E-15 22.64 1.1E-15 21.91 6.1E-16 22.77 6.4E-10 14.63 2.6E-16 23.30 2.4E-14 19.92 2.5E-15 22.04 3.9E-14 21.05 Denaturant Response and Movement of the Transition State. The O 2 6 , Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϫ 1 H

s gradient of each arm of the chevron plot in Fig. 1A reflects the u k dependence of kf or ku on denaturant concentration. Fig. 2A using extrapolated chevron plot and equilibrium data, respectively. shows that for destabilizing mutations these gradients, or m 12 H O values, are linear with ⌬Gu 2 . The absolute changes in mTS-U Ϫ 1 0.25 9.27E-16 0.30 5.7E-14 0.37 1.12E-15 0.23 1.15E-15 0.34 1.46E-15 0.63 3.3E-10 0.34 9.22E-16 0.17 5.74E-16 0.41 3.82E-15 0.70 7.86E-15

*, and mTS-F (the gradients for kf and ku, respectively) are similar using data at 0.401 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ and opposite in sign, although the relative change in m is

TS-U TS-U 12 m calculated from Eqs. greater. The overall m value for unfolding, mU-F can be calcu- kcal ⅐ mol Ϫ 2.09 Ϫ 1.07 Ϫ 0.17 Ϫ 0.54 Ϫ 0.05 Ϫ 1.04 Ϫ 0.06 Ϫ 1.42 Ϫ 0.22 are the change in total free energy of unfolding determined from kinetic chevron plot ( lated from the sum of m and m (Eq. 8). Thus, these

chev TS-U TS-F eqm U-F

u folding and unfolding m values make almost equal contributions 11 . , calculated from Eq. m

O H O 0.07** 0.020.02 0.07 0.01 0.01 0.01 0.04 0.01 0.05 0.02 2 to the change in the overall mU-F with ⌬Gu 2 . This latter change Ϫ 1 H . ⌬⌬ G s f Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ eqm F and k in mU-F (see Fig. 2A, Inset) reflects the dependence of ⌬Gu on 14 ⌽

and ␹ ⌬ 0.04 0.10 TS-F

from Eq. SDS and shows that there is a smaller variation of Gu with SDS calculated from Eq. and ,m chev for destabilizing mutations than for WT. u TS-U †† †† 0.401 chev ␤ TS-U m m F The ratio / for each mutant gives , a measure of the F

from Eq. TS-U U-F well to single exponential function, giving rise to a larger error in T47A and I52A show unusual kinetics and are not analyzed further. ⌽ ⌬⌬ G ⌬⌬ G ⌽ ␤ Table 1. Summary of kinetic data Mutant WT 0.31 * m † ‡ § ¶ ʈ **All errors are either standard errors from curve ﬁtting or from†† propagation. Raw kinetic data of the most destabilizing mutation T46A had lower signal-to-noise than the other mutants and folding data ﬁt less I52A D36A 0.03 F54A 0.06 K41A 0.04 M60A 0.12 F42A 0.04 Y43A 0.13 T46A 0.03 T47A location of the transition state along the reaction coordinate

Curnow and Booth PNAS ͉ January 20, 2009 ͉ vol. 106 ͉ no. 3 ͉ 775 Downloaded by guest on October 4, 2021 relative to the native and unfolded states (4, 15, 16). We previously reported that ␤ is low for WT bR (10), implying that the transition state is close to the SDS-unfolded state. This is not surprising given the amount of residual structure retained in SDS. Here, we find that ␤ shows a linear decrease with increasing ⌬⌬GTS-U, the change in the activation energy of folding (Fig. 2B). Thus, destabilizing mutations cause an apparent movement of the transition state toward the SDS-unfolded state. This movement is correlated with slower refolding rates. Accordingly, we see an approximately equal and opposite trend in the relationship of the activation energy of unfolding, ⌬⌬GTS-F,to mTS-F/mU-F, although the errors are much larger because of the extrapolation of the chevron plot (data not shown).

⌽-Value Analysis. Folding ⌽ values were determined for each mutant and were in good agreement whether the denominator H O chev ⌬⌬Gu 2 was calculated from chevron plot data (⌽F )or eqm derived from equilibrium experiments (⌽F ) (Table 1). ⌽ values using folding and unfolding data were closely correlated (Eq. 13, Fig. S2). To avoid errors arising from extrapolation, we also calculated ⌽ values using ⌬⌬GTS-U and ⌬⌬Gu at a non-zero denaturant concentrations (0.401 and 0.431 ␹SDS, Table 1). These latter ⌽ values closely matched those determined for zero denaturant, with the exception of mutant F42A. Fig. 3 shows ⌽ values for the helix B mutants mapped onto the native structure of bR. Most of the ⌽ values are grouped close to one (Ͼ0.8), suggesting native energetics and interactions of much of this helix in the transition state. However, mutants Y43A and T46A have lower ⌽ values of 0.3 and 0.5, respectively. Such intermediate ⌽ values are less straightforward to interpret, especially for polar to nonpolar amino acid changes as seen here (5). Discussion There is a dearth of information on the folding mechanisms of integral membrane proteins. We show here that it is possible to apply one of the most powerful analytical folding methods to obtain mechanistic detail for a helical membrane protein. Com- bined kinetic, thermodynamic, and mutagenesis studies in the formofa⌽-value analysis have enabled us to begin mapping out the structure and energetics of the transition state for the major, final folding step of bacteriorhodopsin. This type of analysis has been applied to many water-soluble proteins, but we have used it for membrane protein folding. We find several differences in the folding of bR when compared with water-soluble proteins, notably in the denaturant response and the position of the transition state with respect to the folded state.

Solvent Interactions and Structured Unfolded States. There are important distinctions between the folding of membrane proteins and water-soluble proteins both in the nature of the experimental systems and the resulting data. The solvent environment and denaturant interactions are significantly different; while water-soluble proteins are surrounded by a homogenous aqueous environment, bR is solubilized in mixed lipid/detergent micelles in water. Thus, there are interactions of the transmembrane domains of bR with the hydrophobic chains and polar head groups of lipid and detergents as well as of the extrinsic, interhelical loops of the protein with water. The result is a complex array of protein-solvent interactions involving surface amino acid residues that are polar, apolar, and charged. A small chaotrope such as urea gives relatively unstructured states for water-soluble proteins. In contrast, the detergent SDS is used here, resulting in only partial denaturation and the interactions between SDS and bR during unfolding are poorly understood Fig. 3. Structural context of ⌽ values. (A) most mutations give ⌽Ͼ0.8 (deep (17). SDS is an anionic surfactant with a long hydrocarbon tail red) but values for Y43A and T46A are lower (0.3 and 0.5, respectively). (B) and is a bulky denaturant with physical properties that are quite close up of the boxed region from (A) viewed from above shows Y43A packing different from those of urea. against helices A and G and T46A facing helix G. Fig. 3 constructed from PDB The precise structure of the SDS-unfolded state is unclear. It entry 1C3W using Pymol (38).

776 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806953106 Curnow and Booth Downloaded by guest on October 4, 2021 cannot bind the retinal cofactor and lacks native tertiary inter- molecule force spectroscopy study (26) in which Hammond actions (10) but is more structured than the denatured states of behavior was observed with a different set of bR mutants. We most water-soluble proteins. Circular dichroism (CD) spectros- attribute this disparity to the different unfolding methods used copy has shown that the SDS state has an ␣-helical content for force and chemical unfolding. equivalent to approximately 4 transmembrane helices (13) with A possible contribution to the apparent transition state move- a mean residue ellipticity at 226 nm for the folded and unfolded ment reported here is perturbation of the SDS-unfolded state by states of Ϫ22 ϫ 10Ϫ3 and Ϫ17 ϫ 10Ϫ3 deg.cm2.dmolϪ1, respec- mutation, as this unfolded state has a relatively high degree of tively (10) (compared with less than Ϫ4 ϫ 10Ϫ3 deg.cm2.dmolϪ1 structure and mutations can cause changes in the solvation for a random coil). This reduction in helix in SDS could arise energy of this SDS state (27). The changes in m values upon from fraying of some helix ends, or from losses of some mutation (Fig. 2A) are consistent with unfolded state effects. transmembrane helices, probably at the C-terminal end of the Destabilizing mutations decrease the overall m value for the protein (13, 18, 19). The partly helical bO thus provides a unfolding reaction, mU-F, and cause a relatively large increase the different reference position to the more extensively unfolded folding m value, mTS-F. This could partly arise from altered forms of water-soluble proteins, but nonetheless enables the exam- energy and interactions in the SDS-unfolded state of the mutants ination of helix–helix interactions within a two-state system. giving a smaller dependence of unfolding free energy on SDS. SDS is expected to affect bR by changing the physical prop- However, this effect is relatively small; mU-F values are between erties of the DMPC/CHAPS micelle (because SDS will incor- 87–96% of the WT value and both mTS-U and mTS-F contribute porate, forming DMPC/CHAPS/SDS mixed micelles) as well as to the change in mU-F for bR (28). by direct protein-SDS interactions. Although these solvent properties mean a smaller denaturant concentration range is acces- ⌽ Values and Structure in the Transition State. We observe high ⌽ sible by experiment for bR than for water-soluble proteins, linear values for much of helix B (Fig. 3 and Table 1), which are relationships in the form of chevron plots are observed for the consistent with previous suggestions (18, 29, 30) that most of this folding and unfolding kinetics of the mutants studied here (see particular helix, and its structural interactions, are formed early Fig. 1A). By analogy with water-soluble protein studies, these in folding. However, the complexity of the bR refolding system linear relationships suggest that stability correlates with the gives rise to some caveats. ⌽ values are easiest to interpret if the relative degree of denaturant-accessible surface area. Indeed, a free energy of the unfolded state is unaffected by mutation, but direct correlation between the change in unfolding free energy as discussed above, there are likely to be changes in the SDS- and buried surface area has previously been noted for bR (11). unfolded state energy for bR. Such changes in the SDS-bR-state BIOPHYSICS However, the change in unfolding free energy with SDS will also free energy also account for the dependence of the m values H20 reflect alterations in the properties of the DMPC/CHAPS/SDS mTS-U and mU-F on ⌬GU . As discussed by Fersht et al. (5), even micelles that destabilize the protein. Such micelle properties if there are changes in the unfolded state free-energy ⌽ values include micelle size, rigidity, and the lateral pressure imposed on near zero or one are often reliable because the changes in the the protein by the lipid/detergent chains (20, 21). The linear unfolded and transition states induced by the mutation often relationships observed here give a description of denaturant cancel out. Most of our ⌽ values are close to one and the simplest behavior in a micelle system and further emphasize the exper- interpretation of these values as presented here assumes any imental validity of using SDS as a denaturant in kinetic folding changes in unfolded state energy cancel. We also find similar ⌽ experiments on helical membrane proteins. values by extrapolation to zero denaturant as well as at particular SDS concentrations. However, the relatively limited range of Position of the Transition State on the Reaction Coordinate. The denaturant concentrations that can be used in these bR studies chevron plots of bR exemplified in Fig. 1A are more asymmetric means values determined near the midpoint of the kinetic than those obtained for most water-soluble proteins (22), with chevron plot are less reliable. the dependency of kf being rather shallow and that of ku being We observe intermediate ⌽ values at two positions, Y43 and much steeper. This asymmetry reflects a different response to T46, toward the cytoplasmic end of the helix. There are several the denaturant during folding of the membrane protein com- possible explanations for intermediate ⌽ values, including un- pared with a water-soluble protein. The relative gradients of the folded state effects and partial structure formation in the dependence of kf and ku on SDS give a low ␤ value of approx- transition state (although the magnitude of ⌽ is not linear with imately 0.1, indicating that the transition state for bR is globally the extent of structure). They can also arise from other situa- closer to the SDS-unfolded state than the folded state. Higher ␤ tions, such as an ensemble average of two discrete transition values of 0.6–0.9 are generally seen for water-soluble proteins state populations with high and low values. (23), showing that their transition states are closer to their folded Closer inspection of the region with fractional ⌽ values (see states. However, because the unfolded state for bR in SDS is Fig. 3B) shows that Y43 packs against helices A and G and T46 structured, the transition state for this final folding step of bR is packs against helix G. Intriguingly, the light-induced form of bO also significantly structured. These findings agree with earlier (similar to the N intermediate of the photocycle) is characterized reports on the importance of SDS in retaining a critical helical by movements of helices E–G (31–33). In particular, the tops of core for successful folding (13, 18, 24). helices F and G are displaced by several angstroms, with helix F The mutant bR proteins also have low ␤ values and transition tilting away from the center of the protein. A similar change may states close to the partly structured SDS-unfolded state, but take place when the protein is denatured with SDS so that there is an apparent movement of the transition state toward the tertiary interactions between the tops of helices B, F, and G are SDS state with progressive destabilization of the folded state. only partially formed in the folding transition state (which would The Hammond postulate (25) predicts that two consecutive give rise to intermediate ⌽ values). D36, however, has a high ⌽ states in a reaction, such as the unfolded and transition states, value of 0.9, implying native interactions at the cytoplasmic end which have similar energies will have similar structures. Several of the helix. This is an exciting area for further investigation as water-soluble proteins exhibit Hammond behavior (15, 16) with we work toward a complete description of the transition state. destabilizing mutations moving the transition state toward the folded state and reducing the energy difference between the two Conclusions states. We observe the converse for bR; the folding activation It has been unclear how easily the experimental methods devel- energy increases despite the transition state approaching the oped for small water-soluble proteins will translate to integral denatured state. Our results also contrast with a recent single- membrane proteins. Here, we show that a classical approach

Curnow and Booth PNAS ͉ January 20, 2009 ͉ vol. 106 ͉ no. 3 ͉ 777 Downloaded by guest on October 4, 2021 using linear free-energy relationships and site-directed mutagen- mixed micelles of DMPC/CHAPS and reversibly unfolded by the addition or esis can provide unprecedented insight into the folding transition removal of SDS. The kinetics of this process were determined in a stopped-flow state of bR. The free energy surface of folding can be perturbed apparatus by monitoring changes in the retinal chromophore absorbance by mutation and transition state structure inferred from canon- band at 560 nm. All mutants except I52A and T47A showed folding and ical ⌽ values. However, we also find that the solvent and unfolding kinetics similar to WT bR, with a single kinetic phase dominating denaturant interactions are complex and may be quite different folding and unfolding. This was further confirmed by using a photodiode to those during folding of water-soluble proteins. Nonetheless, array to observe simultaneous changes in absorbance at multiple wave- lengths. Typically, the stopped flow was configured with a 2-mm pathlength harnessing the potential of established methods to provide a full ␮ description of the folding pathway now appears to be a realistic and 2.3-nm bandwidth and protein was at 6 M. The refolding yields of all mutants were determined from the regeneration of the native chromophore goal for helical membrane proteins. The excellent agreement using a CARY UV-Vis spectrophotometer and were comparable to WT yields. between equilibrium and kinetically derived parameters shown Graphs were prepared using Origin (Microcal, Inc.) or GraFit (Erithacus Software). here, and the linear free-energy relationships in both datasets, is The procedure for determining ⌽ values (see SI Appendix) has been exten- especially valuable and paves the way for further detailed studies sively described by Fersht and colleagues (22). Briefly, kinetic and equilibrium in this field. data were used to determine the effects of point mutations on the total free H O energy change upon unfolding (⌬G 2 ) and the activation energy of folding Methods u (⌬⌬GTS-U) (Eq. 6, SI). The parameter ␤ is the ratio of the m value for folding Expression of bR Mutants. bR mutants in plasmid pMPK85 (11, 34) were a kind (mTS-U) to the overall m value (mU-F), where the m values reflect the denaturant gift from J. U. Bowie (University of California, Los Angeles, CA). H. salinarum dependence of the activation free energy for folding and the overall free strain L33 (which does not express WT bR) was transformed with pMPK85 energy of the unfolding reaction, respectively. according to the protocol of Cline and Doolittle (35) and transformants were selected and maintained using mevinolin (Sigma) at 10 ␮M in media plates and ACKNOWLEDGMENTS. We thank Duan Yang for mutant plasmids and help broth. Growth of recombinant cells and purification of recombinant bR was with H. salinarum transformations, Kathleen Moreton for preparing mutant according to Oesterhelt and Stoeckenius (36). All mutants were characterized protein, Jim Bowie for useful discussions and provision of plasmids and by mass spectrometry (37). protein, and Sophie Jackson for a critique of the manuscript and analyses. We acknowledge funding from the BBSRC (BB/D001676). P.J.B. holds a Royal Data Collection and Analyses. The collection and analysis of kinetic data has Society-Wolfson Research Merit Award and is a member of the E-MeP EU previously been described in detail (10). Briefly, the protein was maintained in consortium.

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