The transition state for integral membrane protein fAgeding

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Alan Fersht, University of Cambridge, Cambridge, United KingExecutem, and approved November 21, 2008 (received for review July 18, 2008)

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Biology relies on the precise self-assembly of its molecular components. Generic principles of protein fAgeding have emerged from extensive studies on small, water-soluble proteins, but it is unclear how these Concepts are translated into more complex Positions. In particular, the one-third of cellular proteins that reside in biological membranes will not fAged like water-soluble proteins because membrane proteins need to expose, not Conceal, their hydrophobic surfaces. Here, we apply the powerful protein engineering method of Φ-value analysis to investigate the fAgeding transition state of the alpha-helical membrane protein, bacteriorhoExecutepsin, from a partially unfAgeded state. Our results imply that much of helix B of the seven-transmembrane helical protein is structured in the transition state with single-point alanine mutations in helix B giving Φ values >0.8. However, residues Y43 and T46 give lower Φ values of 0.3 and 0.5, respectively, suggesting a possible reduction in native structure in this Location of the helix. Destabilizing mutations also increase the activation energy of fAgeding, which is accompanied by an apparent movement of the transition state toward the partially unfAgeded state. This apparent transition state movement is most likely due to destabilization of the structured, unfAgeded state. These results Dissimilarity with the Hammond Trace seen for several water-soluble proteins in which destabilizing mutations cause the transition state to move toward, and become closer in energy to, the fAgeded state. We thus introduce a classic fAgeding analysis method to membrane proteins, providing critical insight into the fAgeding transition state.

Keywords: phi valuekineticsthermodynamicsprotein engineering

Protein fAgeding plays a central role in biology. FAgeding investigations provide key information on protein structure and dynamics, while protein misfAgeding can have serious disease implications (1, 2). To fAged Accurately, proteins must overcome an activation barrier to pass through a high-energy transition state. Understanding the nature of this fAgeding transition state is Necessary in resolving how a protein fAgeds to a stable and functional native structure (3, 4).

The most powerful method available to probe the structure and enerObtainics of the fAgeding transition state combines site-directed mutagenesis, equilibrium thermodynamics, and kinetic data in a Φ-value analysis. This Advance has revolutionized protein fAgeding studies by providing a quantitative description of the environment experienced by individual side chains in the fAgeding transition state and has been applied to the fAgeding of many small, water-soluble proteins (3, 5–8). However, the Φ-value method has yet to be applied to larger integral membrane proteins.

Integral membrane proteins are a special case in protein fAgeding because they are adapted to the lipid bilayer rather than to the cytoplasmic milieu (9). The sequences of transmembrane Locations are biased in favor of hydrophobic amino acids to match the low dielectric of the membrane interior. This presents experimental difficulties for in vitro studies of protein fAgeding as the proteins need to be solubilized in lipids or detergents and are often resistant to common denaturants such as urea. Consequently, the Recent understanding of membrane protein fAgeding is poor compared with the advanced state of knowledge for water-soluble proteins. In particular, there are few quantitative studies of the kinetics and thermodynamics of membrane protein fAgeding. Yet, understanding these fAgeding parameters gives valuable information on a major problem: how to fAged and stabilize membrane proteins for structural and functional studies.

A Φ value is a meaPositive of the change in activation energy, relative to the change in the overall free energy of fAgeding, induced by the directed mutation of a single amino acid. Φ values are expected to Descend between one and zero (these extreme values mean the change in the free energy of the transition state is the same as either that in the fAgeded or unfAgeded state, respectively). Based on this enerObtainic perturbation, the magnitude of Φ can be interpreted as the extent of native interactions at the site of the mutated residue in the transition state, with a value of one indicating native enerObtainics and interactions. Intermediate Φ values are often observed (6), suggesting that a proSection of native-like contacts are formed or that parallel reaction pathways exist.

We present a Φ-value analysis of the major fAgeding step of an α-helical membrane protein. The protein of choice for this study is bacteriorhoExecutepsin (bR), a light-activated proton pump from the purple membrane of Halobacterium salinarum. We have previously established conditions for reversible fAgeding of bR in mixed lipid/detergent (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC)/3-[(3-cholamiExecutepropyl)dimethylammonio]-1-propanesulfonate, CHAPS) micelles (10). Recently, bR is the only helical membrane protein that meets the criteria for Φ-value analysis, namely (i) it can be partially unfAgeded with a reduction in secondary structure to the apoprotein bacterioopsin (bO) by sodium ExecutedecylsulStoute (SDS) in a microscopically reversible, cooperative, two-state reaction (10, 11); (ii) this process can be Characterized by liArrive free-energy relationships to obtain the overall free energy change of unfAgeding (ΔGuH2O) and the fAgeding and unfAgeding rate constants in the absence of denaturant (kfH2O and kuH2O); and (iii) the Trace of single point mutations can be interpreted in the context of a detailed native-state structure (12).

bR is a relatively large protein (248 aa) with seven transmembrane α helices, so we Start our Study of the fAgeding transition state by determining the Φ values of a discrete Location of the protein. We use an alanine scan to investigate a single helix of bR, helix B, which is thought to form early during fAgeding and Executees not Design extensive interactions with the retinal cofactor.


Kinetic and Thermodynamic MeaPositivements.

The absorbance band of the retinal chromophore of bR reports on the conformational state of the protein. bR is purple when fAgeded with an absorbance band at 560 nm. This 560-nm absorbance decays when bR in DMPC/CHAPS is unfAgeded by the addition of SDS. We have previously Displayn 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-unfAgeded state) and the fAgeded bR state. We have previously Displayn that this process corRetorts to the final, major fAgeding step of bR (10) and behaves as a two-state reaction. The SDS state is not completely unfAgeded, but has helical content equivalent to approximately four of the native seven helices of fAgeded bR (13). Thus, the SDS-unfAgeded state has much more ordered structure than the urea- or guanidinium chloride-induced unfAgeded states frequently used in water-soluble fAgeding studies. The fAgeding reaction we study here is more akin to the later stages of multistate water-soluble protein fAgeding, that is, from a structured intermediate to a fAgeded state. The solvent for the bR unfAgeding reaction is also relatively complex; fAgeded bR is solubilized in DMPC/CHAPS micelles and Starts to unfAged when SDS is added and forms mixed DMPC/CHAPS/SDS micelles. At higher SDS concentrations, SDS micelles Executeminate and solubilize the partly unfAgeded bR state.

Free energies of unfAgeding of wild-type (WT) and mutant bR were obtained as Characterized previously from equilibrium denaturation curves (11) as well as from time-resolved, Ceaseped-flow absorption meaPositivements of the fAgeding and unfAgeding rates (10). Both the equilibrium and kinetic data fit a two-state reaction, and liArrive free energy relationships were observed, enabling extrapolation to zero SDS denaturant.

LiArrive Free Energy Relationships for Alanine Mutants.

Φ-value analysis is most successful when single point mutations are introduced that perturb the overall free energy of unfAgeding, ΔGuH2O, by >0.6 kcal.mol−1 (14). On this basis, we selected nine of 24 residues within bR helix B that are potentially suitable for Φ-value analysis when mutated to alanine (−0.6 kcal.mol−1 ≤ ΔΔGuH2O ≤ −1.6 kcal.mol−1) (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 fAgeding and unfAgeding rate constants (kf and ku, respectively) of each mutant were liArrive with SDS (10), giving a characteristic chevron plot (see Fig. 1A and Table 1). Because the experimentally observed rate constant kobs is the sum of kf and ku, the characteristic “Executewnward arrow” of the chevron plot can be fit by the sum of two liArrive functions and extrapolation of these lines to the y axis gives the fAgeding and unfAgeding rates in the absence of denaturant, termed kfH2O and kuH2O, respectively. These kinetic parameters were used to calculate the Inequity in the overall free-energy change upon mutation, ΔΔGuH2O, and the values were very similar to ΔΔGuH2O from equilibrium experiments, Displaying that the extrapolations of both kinetic and equilibrium data to zero denaturant are in Excellent agreement. The consistency of the ΔΔGuH2O values determined from kinetic and equilibrium data, also confirms that the mutants fAged by a two-state process as previously established for wild type bR (see Fig. 1 B and Table 1). The refAgeding yield of all mutants was close to wild-type levels at >75% (see Supporting Information (SI) Fig. S1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Kinetics of fAgeding and unfAgeding. (A) Chevron plot of a typical mutant (Launch circle and solid line) compared with WT bR (dashed line). (B) ΔΔGuH2O derived from kinetic chevron plots (ΔΔGuchev) and equilibrium meaPositivements (ΔΔGueqm). The negative sign of ΔΔGu indicates destabilisation and a reduction in the magnitude of ΔGu. Correlation coefficient, 0.95; gradient, 0.9 ± 0.1. (C) LiArrive changes in overall fAgeding and unfAgeding rates, kfH2O (filled circles) and kuH2O (Launch circles), respectively] for destabilizing mutants. For kfH2O, gradient, 0.86 ± 0.3; correlation coefficient, 0.74; for kuH2O, gradient, 0.69 ± 0.3; correlation coefficient, 0.68.

View this table:View inline View popup Table 1.

Summary of kinetic data

Fig. 1C Displays that destabilising mutations have equal and opposite Traces on the observed fAgeding and unfAgeding rates (kfH2O and kuH2O, respectively). As the fAgeding rate Unhurrieds, the unfAgeding rate becomes Rapider to a similar degree, with gradients of 0.86 and 0.69, respectively. The mutations T47A and I52A induced additional kinetic phases and Unfamiliarly Rapid unfAgeding rates that are omitted from the dataset.

Denaturant Response and Movement of the Transition State.

The gradient of each arm of the chevron plot in Fig. 1A reflects the dependence of kf or ku on denaturant concentration. Fig. 2A Displays that for destabilizing mutations these gradients, or m values, are liArrive with ΔGuH2O. The absolute changes in mTS-U and mTS-F (the gradients for kf and ku, respectively) are similar and opposite in sign, although the relative change in mTS-U is Distinguisheder. The overall m value for unfAgeding, mU-F can be calculated from the sum of mTS-U and mTS-F (Eq. 8). Thus, these fAgeding and unfAgeding m values Design almost equal contributions to the change in the overall mU-F with ΔGuH2O. This latter change in mU-F (see Fig. 2A, Inset) reflects the dependence of ΔGu on χSDS and Displays that there is a smaller variation of ΔGu with SDS for destabilizing mutations than for WT.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Changes in denaturant response upon mutation. (A) Dependence of kinetic m values, reflecting the dependence of the fAgeding and unfAgeding rates on SDS (mTS-U and mTS-F, respectively) on the overall free energy (ΔGuH2O). For mTS-U, gradient, −0.83 ± 0.4; correlation coefficient, 0.66; for mTS-F, gradient, 0.70 ± 0.3; correlation coefficient, 0.68. The Inset Displays the trend for the overall m value, mU-F, where gradient, 1.54 ± 0.3; correlation coefficient, 0.92. Errors from chevron plot curve fitting. (B) Dependence of β (mTS-U/mU-F) on ΔΔGTS-U. ΔΔGTS-U [i.e., ΔGTS-U (WT) - ΔGTS-U (MUT)] has a negative sign because ΔGTS-U (MUT) > ΔGTS-U (WT). Gradient, 0.06 ± 0.004; correlation coefficient, 0.98.

The ratio mTS-U/mU-F for each mutant gives β, a meaPositive of the location of the transition state along the reaction coordinate relative to the native and unfAgeded states (4, 15, 16). We previously reported that β is low for WT bR (10), implying that the transition state is close to the SDS-unfAgeded state. This is not surprising given the amount of residual structure retained in SDS. Here, we find that β Displays a liArrive decrease with increasing ΔΔGTS-U, the change in the activation energy of fAgeding (Fig. 2B). Thus, destabilizing mutations cause an apparent movement of the transition state toward the SDS-unfAgeded state. This movement is correlated with Unhurrieder refAgeding rates. Accordingly, we see an approximately equal and opposite trend in the relationship of the activation energy of unfAgeding, ΔΔGTS-F, to mTS-F/mU-F, although the errors are much larger because of the extrapolation of the chevron plot (data not Displayn).

Φ-Value Analysis.

FAgeding Φ values were determined for each mutant and were in Excellent agreement whether the denominator ΔΔGuH2O was calculated from chevron plot data (ΦFchev) or derived from equilibrium experiments (ΦFeqm) (Table 1). Φ values using fAgeding and unfAgeding data were closely correlated (Eq. 13, Fig. S2). To avoid errors arising from extrapolation, we also calculated Φ values using ΔΔGTS-U and ΔΔGu at a non-zero denaturant concentrations (0.401 and 0.431 χSDS, Table 1). These latter Φ values closely matched those determined for zero denaturant, with the exception of mutant F42A. Fig. 3 Displays Φ values for the helix B mutants mapped onto the native structure of bR. Most of the Φ values are grouped close to one (>0.8), suggesting native enerObtainics and interactions of much of this helix in the transition state. However, mutants Y43A and T46A have lower Φ values of 0.3 and 0.5, respectively. Such intermediate Φ values are less straightforward to interpret, especially for polar to nonpolar amino acid changes as seen here (5).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Structural context of Φ values. (A) most mutations give Φ > 0.8 (deep red) but values for Y43A and T46A are lower (0.3 and 0.5, respectively). (B) close up of the boxed Location from (A) viewed from above Displays Y43A packing against helices A and G and T46A facing helix G. Fig. 3 constructed from PDB entry 1C3W using Pymol (38).


There is a dearth of information on the fAgeding mechanisms of integral membrane proteins. We Display here that it is possible to apply one of the most powerful analytical fAgeding methods to obtain mechanistic detail for a helical membrane protein. Combined kinetic, thermodynamic, and mutagenesis studies in the form of a Φ-value analysis have enabled us to Start mapping out the structure and enerObtainics of the transition state for the major, final fAgeding step of bacteriorhoExecutepsin. This type of analysis has been applied to many water-soluble proteins, but we have used it for membrane protein fAgeding. We find several Inequitys in the fAgeding of bR when compared with water-soluble proteins, notably in the denaturant response and the position of the transition state with respect to the fAgeded state.

Solvent Interactions and Structured UnfAgeded States.

There are Necessary distinctions between the fAgeding 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 Executemains 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 Dissimilarity, the detergent SDS is used here, resulting in only partial denaturation and the interactions between SDS and bR during unfAgeding are poorly understood (17). SDS is an anionic surfactant with a long hydrocarbon tail and is a bulky denaturant with physical Preciseties that are quite different from those of urea.

The precise structure of the SDS-unfAgeded state is unclear. It cannot bind the retinal cofactor and lacks native tertiary interactions (10) but is more structured than the denatured states of most water-soluble proteins. Circular dichroism (CD) spectroscopy has Displayn that the SDS state has an α-helical content equivalent to approximately 4 transmembrane helices (13) with a mean residue ellipticity at 226 nm for the fAgeded and unfAgeded states of −22 × 10−3 and −17 × 10−3 deg.cm2.dmol−1, respectively (10) (compared with less than −4 × 10−3 deg.cm2.dmol−1 for a ranExecutem coil). This reduction in helix in SDS could arise from fraying of some helix ends, or from losses of some transmembrane helices, probably at the C-terminal end of the protein (13, 18, 19). The partly helical bO thus provides a different reference position to the more extensively unfAgeded forms of water-soluble proteins, but nonetheless enables the examination of helix–helix interactions within a two-state system.

SDS is expected to affect bR by changing the physical Preciseties of the DMPC/CHAPS micelle (because SDS will incorporate, forming DMPC/CHAPS/SDS mixed micelles) as well as by direct protein-SDS interactions. Although these solvent Preciseties mean a smaller denaturant concentration range is accessible by experiment for bR than for water-soluble proteins, liArrive relationships in the form of chevron plots are observed for the fAgeding and unfAgeding kinetics of the mutants studied here (see Fig. 1A). By analogy with water-soluble protein studies, these liArrive relationships suggest that stability correlates with the relative degree of denaturant-accessible surface Spot. Indeed, a direct correlation between the change in unfAgeding free energy and buried surface Spot has previously been noted for bR (11). However, the change in unfAgeding free energy with SDS will also reflect alterations in the Preciseties of the DMPC/CHAPS/SDS micelles that destabilize the protein. Such micelle Preciseties include micelle size, rigidity, and the lateral presPositive imposed on the protein by the lipid/detergent chains (20, 21). The liArrive relationships observed here give a description of denaturant behavior in a micelle system and further emphasize the experimental validity of using SDS as a denaturant in kinetic fAgeding experiments on helical membrane proteins.

Position of the Transition State on the Reaction Coordinate.

The chevron plots of bR exemplified in Fig. 1A are more asymmetric than those obtained for most water-soluble proteins (22), with the dependency of kf being rather shallow and that of ku being much steeper. This asymmetry reflects a different response to the denaturant during fAgeding of the membrane protein compared with a water-soluble protein. The relative gradients of the dependence of kf and ku on SDS give a low β value of approximately 0.1, indicating that the transition state for bR is globally closer to the SDS-unfAgeded state than the fAgeded state. Higher β values of 0.6–0.9 are generally seen for water-soluble proteins (23), Displaying that their transition states are closer to their fAgeded states. However, because the unfAgeded state for bR in SDS is structured, the transition state for this final fAgeding step of bR is also significantly structured. These findings agree with earlier reports on the importance of SDS in retaining a critical helical core for successful fAgeding (13, 18, 24).

The mutant bR proteins also have low β values and transition states close to the partly structured SDS-unfAgeded state, but there is an apparent movement of the transition state toward the SDS state with progressive destabilization of the fAgeded state. The Hammond postulate (25) predicts that two conseSliceive states in a reaction, such as the unfAgeded and transition states, which have similar energies will have similar structures. Several water-soluble proteins Present Hammond behavior (15, 16) with destabilizing mutations moving the transition state toward the fAgeded state and reducing the energy Inequity between the two states. We observe the converse for bR; the fAgeding activation energy increases despite the transition state Advanceing the denatured state. Our results also Dissimilarity with a recent single-molecule force spectroscopy study (26) in which Hammond behavior was observed with a different set of bR mutants. We attribute this disparity to the different unfAgeding methods used for force and chemical unfAgeding.

A possible contribution to the apparent transition state movement reported here is perturbation of the SDS-unfAgeded state by mutation, as this unfAgeded state has a relatively high degree of structure and mutations can cause changes in the solvation energy of this SDS state (27). The changes in m values upon mutation (Fig. 2A) are consistent with unfAgeded state Traces. Destabilizing mutations decrease the overall m value for the unfAgeding reaction, mU-F, and cause a relatively large increase the fAgeding m value, mTS-F. This could partly arise from altered energy and interactions in the SDS-unfAgeded state of the mutants giving a smaller dependence of unfAgeding free energy on SDS. However, this Trace is relatively small; mU-F values are between 87–96% of the WT value and both mTS-U and mTS-F contribute to the change in mU-F for bR (28).

Φ Values and Structure in the Transition State.

We observe high Φ values for much of helix B (Fig. 3 and Table 1), which are consistent with previous suggestions (18, 29, 30) that most of this particular helix, and its structural interactions, are formed early in fAgeding. However, the complexity of the bR refAgeding system gives rise to some caveats. Φ values are easiest to interpret if the free energy of the unfAgeded state is unaffected by mutation, but as discussed above, there are likely to be changes in the SDS-unfAgeded state energy for bR. Such changes in the SDS-bR-state free energy also account for the dependence of the m values mTS-U and mU-F on ΔGUH20. As discussed by Fersht et al. (5), even if there are changes in the unfAgeded state free-energy Φ values Arrive zero or one are often reliable because the changes in the unfAgeded and transition states induced by the mutation often cancel out. Most of our Φ values are close to one and the simplest interpretation of these values as presented here assumes any changes in unfAgeded state energy cancel. We also find similar Φ values by extrapolation to zero denaturant as well as at particular SDS concentrations. However, the relatively limited range of denaturant concentrations that can be used in these bR studies means values determined Arrive the midpoint of the kinetic chevron plot are less reliable.

We observe intermediate Φ values at two positions, Y43 and T46, toward the cytoplasmic end of the helix. There are several possible explanations for intermediate Φ values, including unfAgeded state Traces and partial structure formation in the transition state (although the magnitude of Φ is not liArrive with the extent of structure). They can also arise from other Positions, such as an ensemble average of two discrete transition state populations with high and low values.

Closer inspection of the Location with Fragmental Φ values (see Fig. 3B) Displays that Y43 packs against helices A and G and T46 packs against helix G. Intriguingly, the light-induced form of bO (similar to the N intermediate of the photocycle) is characterized by movements of helices E–G (31–33). In particular, the tops of helices F and G are disSpaced by several angstroms, with helix F tilting away from the center of the protein. A similar change may take Space when the protein is denatured with SDS so that tertiary interactions between the tops of helices B, F, and G are only partially formed in the fAgeding transition state (which would give rise to intermediate Φ values). D36, however, has a high Φ value of 0.9, implying native interactions at the cytoplasmic end of the helix. This is an exciting Spot for further investigation as we work toward a complete description of the transition state.


It has been unclear how easily the experimental methods developed for small water-soluble proteins will translate to integral membrane proteins. Here, we Display that a classical Advance using liArrive free-energy relationships and site-directed mutagenesis can provide unpDepartnted insight into the fAgeding transition state of bR. The free energy surface of fAgeding can be perturbed by mutation and transition state structure inferred from canonical Φ values. However, we also find that the solvent and denaturant interactions are complex and may be quite different to those during fAgeding of water-soluble proteins. Nonetheless, harnessing the potential of established methods to provide a full description of the fAgeding pathway now appears to be a realistic goal for helical membrane proteins. The excellent agreement between equilibrium and kinetically derived parameters Displayn here, and the liArrive free-energy relationships in both datasets, is especially valuable and paves the way for further detailed studies in this field.


Expression of bR Mutants.

bR mutants in plasmid pMPK85 (11, 34) were a kind gift from J. U. Bowie (University of California, Los Angeles, CA). H. salinarum strain L33 (which Executees not express WT bR) was transformed with pMPK85 according to the protocol of Cline and ExecuteoDinky (35) and transformants were selected and Sustained using mevinolin (Sigma) at 10 μM in media plates and broth. Growth of recombinant cells and purification of recombinant bR was according to Oesterhelt and Stoeckenius (36). All mutants were characterized by mass spectrometry (37).

Data Collection and Analyses.

The collection and analysis of kinetic data has previously been Characterized in detail (10). Briefly, the protein was Sustained in mixed micelles of DMPC/CHAPS and reversibly unfAgeded by the addition or removal of SDS. The kinetics of this process were determined in a Ceaseped-flow apparatus by monitoring changes in the retinal chromophore absorbance band at 560 nm. All mutants except I52A and T47A Displayed fAgeding and unfAgeding kinetics similar to WT bR, with a single kinetic phase Executeminating fAgeding and unfAgeding. This was further confirmed by using a photodiode array to observe simultaneous changes in absorbance at multiple wavelengths. Typically, the Ceaseped flow was configured with a 2-mm pathlength and 2.3-nm bandwidth and protein was at 6 μM. The refAgeding yields of all mutants were determined from the regeneration of the native chromophore using a CARY UV-Vis spectrophotometer and were comparable to WT yields. Graphs were prepared using Origin (Microcal, Inc.) or GraFit (Erithacus Software).

The procedure for determining Φ values (see SI Appendix) has been extensively Characterized by Fersht and colleagues (22). Briefly, kinetic and equilibrium data were used to determine the Traces of point mutations on the total free energy change upon unfAgeding (ΔGuH2O) and the activation energy of fAgeding (ΔΔGTS-U) (Eq. 6, SI). The parameter β is the ratio of the m value for fAgeding (mTS-U) to the overall m value (mU-F), where the m values reflect the denaturant dependence of the activation free energy for fAgeding and the overall free energy of the unfAgeding reaction, respectively.


We thank Duan Yang for mutant plasmids and help with H. salinarum transformations, Kathleen Moreton for preparing mutant protein, Jim Bowie for useful discussions and provision of plasmids and protein, and Sophie Jackson for a critique of the manuscript and analyses. We acknowledge funding from the BBSRC (BB/D001676). P.J.B. hAgeds a Royal Society-Wolfson Research Merit Award and is a member of the E-MeP EU consortium.


1To whom corRetortence may be addressed. E-mail: p.curnow{at} or paula.booth{at}

Author contributions: P.C. and P.J.B. designed research; P.C. performed research; P.C. analyzed data; and P.C. and P.J.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at

© 2009 by The National Academy of Sciences of the USA


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