Protein Science ~1999!, 8:913920. Cambridge University Press. Printed in the USA.

Copyright 1999 The Protein Society

Computational studies on mutant protein stability: The correlation between surface thermal expansion and protein stability

ROCIO PALMA 1 and PAUL M.G. CURMI

Initiative in Biomolecular Structure, School of Physics, University of New South Wales, Sydney NSW 2052, Australia ~Received August 5, 1998; Accepted December 29, 1998!

Abstract Thermal stability of mutant proteins has been investigated using temperature dependent molecular dynamics ~MD! simulations in vacuo. The numerical modeling was aimed at mimicking protein expansion upon heating. After the conditions for an expanding protein accessible surface area were established for T4 lysozyme and barnase wild-type proteins, MD simulations were carried out under the same conditions using the crystal structures of several mutant proteins. The computed thermal expansion of the accessible surface area of mutant proteins was found to be strongly correlated with their experimentally measured stabilities. A similar, albeit weaker, correlation was observed for model mutant proteins. This opens the possibility of obtaining stability information directly from protein structure. Keywords: molecular dynamics; protein stability; single-site mutations; thermal expansion

The production of mutant proteins with enhanced thermal stability has become an important area of bioengineering. This area would be enhanced by a quantitative understanding of the effects of specific interactions on a proteins structure and stability. Similarly, protein de novo design and modification would benefit from such an understanding. The systematic experimental study of the effects of amino acid interactions on stability has been made possible by site-directed mutagenesis techniques. Point mutations in particular permit one to remove defined interactions without introducing new or unfavorable interactions. But even chemically simple alterations can produce localized structural shifts, changing many different interactions in the folded protein. While experimental methods such as X-ray crystallography or NMR give information about specific changes in structure due to a mutation, thermodynamic measurements cannot separate the specific factor or factors responsible for stability changes. Additionally, there is no direct relationship between structure and stability changes. Large structural shifts could result in minimal stability changes and vice versa. The flexibility of protein structure results in most of engineered mutations showing only minimal stability changes ~Serrano et al., 1992; Matthews, 1995, 1996; Sauer et al., 1996!. At present, there are only few methods that permit one to analyze the effects of a mutation on the stability of a target protein

Reprint requests to: Paul Curmi, Initiative in Biomolecular Structure, School of Physics, University of New South Wales, Sydney NSW 2052, Australia; e-mail: P.Curmi@unsw.edu.au. 1 Present address: Rocio Palma, Department of Food Science, Cornell University, Ithaca, New York 14853.

based on the knowledge of the changes due to a mutation on its structure ~Gilis & Rooman, 1996, 1997!. Clearly, advances in such methodologies will provide an extremely powerful tool for the establishment of rules for successful protein modification. A natural strategy for analyzing the structure-stability relationship is to examine its behavior in a molecular dynamics ~MD! simulation, where detailed information about protein structure is used. Much has been learned about protein structure and dynamics via such techniques. Limitations in computer speed and the accuracy of the potential functions have largely confined these methods to quasi-equilibrium simulations. Direct MD studies of complete protein denaturation are just beginning to emerge ~Bond et al., 1997; Lazaridis & Karplus, 1997; Tirado-Rives et al., 1997; Li & Daggett, 1998! while reversible folding studies are currently confined to peptides ~Daura et al., 1998!. An alternative MD approach to the study of the effects of mutation on the stability of a protein is the free energy simulation technique ~Gao et al., 1989; Tidor & Karplus, 1991!. In these methods, a thermodynamic cycle is constructed that consists of the folded and unfolded states of the wild-type protein and the mutant protein. By determining the free energy changes that occur on mutation of both the folded and unfolded states, the changes in stability of the mutant protein can be determined. The reliability of these calculations is still the subject of debate ~Yun-yu et al., 1993!. In this work, a more physical approach to gaining information on protein thermodynamics has been used. Since thermal denaturation is a temperature dependent, dynamic process of the transition from the folded to the unfolded state, temperature dependent MD simulations were used to obtain the changes in protein structure as

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a function of temperature. We have focused our attention on the temperature dependence of the solvent accessible surface area ~ASA! of the protein. We have found sets of MD simulation conditions, which result in a thermally expanding wild-type protein. Our results show that mutant protein stability and the surface thermal expansion obtained from MD simulations are well correlated. This permits the prediction of mutant protein thermal stability based solely on the analysis of the dynamics of its structure. To investigate the utility of such an approach, it is essential to study systems that are well characterized both structurally and thermodynamically. We have chosen two proteins: T4 lysozyme and barnase. In each of these systems, a large number of sitedirected mutant proteins have been constructed. The X-ray or NMR structures of these mutant proteins have been determined and their stabilities have been measured ~Serrano et al., 1992; Matthews, 1996!. Thus, for each of these systems, the individual mutant protein structures can be used for MD simulation from which temperature dependent properties can be calculated and compared with the stability parameters. Results and discussion Temperature dependent MD simulations In solution, a protein structure continuously evolves under the influence of changing temperature. To mimic this process in an MD simulation, quasi-equilibrated protein heating was carried out by coupling the protein system to a thermal bath where the temperature of the bath was incremented slowly. To achieve this, a protein structure was allowed to equilibrate at an initial temperature equal to T1 ~K!. The equilibrated structure and the corresponding atomic velocities were used to start a data collection MD simulation where the temperature of the thermal bath was incremented by 58 every N steps until a final temperature of T2 ~K! was reached. To monitor the effects of heating, we analyzed the changes in the proteins ASA as the temperature increased. By measuring the ASA, we are following both the experimentally demonstrated thermal expansion of protein volume ~Bull & Breese, 1973; Frauenfelder et al., 1987; Tilton et al., 1992! and the variations in the roughness of the protein surface. The importance of the proteinsolvent interface as a crucial factor determining protein stability has long been recognized. Protein stability has been shown to be dependent on the net change that occurs in the ASA on moving from the native state to the denatured state ~Tanford, 1970; Chothia, 1974; Eisenberg & McLachlan, 1986; Ooi et al., 1987; Makhatadze & Privalov, 1990; Privalov & Makhatadze, 1990!. However, the net change in ASA resulting from a point mutation is too small to provide a reliable measure of the change in stability due to the mutation. Mimicking proteinsolvent interactions Proteinsolvent interactions play a crucial role on protein thermodynamics, so these interactions should to be included in any model. To simplify our computations ~save CPU time!, no explicit solvent was included in our simulations. As a result, there is an effective surface term due to the protein being in vacuo, which is caused by the net attractive forces between the protein atoms. This would normally cause the protein to contract during simulation. So, to obtain an expanding protein ASA in vacuo is nontrivial.

R. Palma and P.M.G. Curmi To mimic the proteinsolvent interaction and, hence, improve the approximation that our simulation makes to the real situation: ~1! the protein surface charges were neutralized; ~2! only noncore residues were coupled to the thermal bath, and ~3! the frictional coupling parameter to the thermal bath was determined by a linear, decreasing function of temperature. Residue selection for charge neutralization and differential thermal coupling was based on solvent accessibility. The side chains of exposed, charged residues were neutralized while no thermal coupling was applied to buried, core residues. This resulted in three clearly defined regions in the protein: ~1! the surface with thermal coupling but no explicit charges, ~2! the core with full charges but without coupling to the thermal bath, and ~3! in between are residues with their full charges and thermal coupling. With the thermal coupling parameter fbeta, a large value ~ 10 ps 1 ! or a small value ~ 1 ps 1 ! resulted in a protein that contracted with temperature ~as measured via ASA!. By trial and error, we found that for an expanding protein, the coupling parameter should be in the range of 25 ps 1 and should decrease with increasing temperature. Different values of fbeta, including a constant value, were tested on the wild-type proteins. Generally, when fbeta decreases with temperature, the protein MD simulation shows an ASA that increases smoothly with temperature. Typically, the temperature dependent coupling parameter was given by fbeta fbeta0 ~d fbeta0dT !T ~1!

where T ~K! is the simulation temperature. For each wild-type protein, a number of simulations were run differing in: ~1! the intercept ~fbeta0 ! and the slope ~d fbeta0dT ! of the friction coefficient, ~2! the ASA thresholds used to define residue solvent exposure, and ~3! the type of surface residues where charges are neutralized. From MD simulations with wild-type barnase, there seems to be a strong correlation between the amount of charge neutralized at the protein surface and the value for the coupling parameter ~data not shown!. Additionally, the results were found to be dependent on the type of force field used for the simulations, the parameters for the calculation of the nonbonded energy and the starting coordinates. After running numerous MD simulations on the two wild-type proteins, the set of simulation conditions that gave the smoothest, increasing plot of ASA vs. temperature for each protein was selected. These conditions were then applied to the MD simulations of the respective mutant proteins. Protein thermal expansion During the quasi-equilibrium simulations, the various mutant proteins tended to expand steadily with increasing temperature as per the wild-type protein. This is reflected in an increasing ASA obtained for both T4 lysozyme and barnase mutant proteins as a function of temperature ~Fig. 1A,B!. Occasionally, discontinuous jumps in the ASA were observed, presumably due to the sudden shift of a side-chain position. These transitions were most commonly seen in the trajectories of less stable mutant proteins. To keep the analysis simple, these fluctuations were ignored except when they were excessively large ~see Materials and methods!. The ASA as a function of temperature was fit to a first approximation by a linear function of temperature: ASA~T ! ASA8 ~dASA0dT !T ~2!

Correlation between protein expansion and stability

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Fig. 1. Accessible surface areas for ~A! T4-lysozyme and ~B! barnase wild-type proteins and some mutants plotted as a function of temperature. The figure shows the evolution of the ASA as a function of temperature obtained from the MD simulations for the proteins used in this study. In each graph, the fitting line used to calculate a, the surface thermal expansion coefficient, is shown.

where the linear fit was carried out using a least-squares procedure over the temperature range from T1 to T2 ~K!. The first striking result from simulations of mutant proteins was that stable mutant proteins showed a smaller increase in ASA with temperature compared with the wild-type protein. In contrast, unstable ones showed a larger rate of increase of ASA as temperature increases. This result was analytically expressed by fitting the evolution of the ASA with a linear function of temperature and calculating the ratio of the slope to the intercept: ~dASA0dT !0ASA8 ~3!

The surface thermal expansion coefficient in the case of a stable mutant protein was smaller than that of the wild-type protein, and it was larger in the case of the unstable ones. This result pointed to the possibility of a gross classification of mutant proteins as stable or unstable compared to the wild-type.

Correlation between thermal expansion and stability We examined the relationship between the surface thermal expansion coefficient a and the available experimental data on protein stability for the two sets of mutant proteins ~Tables 1, 2!. Figure 2 shows the plot of a vs. the measured unfolding temperature Tm for the wild-type and mutant T4 lysozymes. There is a strong correlation between the measured and computed data with a linear correlation coefficient equal to 0.86. For barnase, the most comprehensive thermodynamic data available for the mutant proteins were the change in Gibbs free energy

where a has the units of the thermal expansion ~K 1 ! and was called the surface thermal expansion coefficient. The values of the surface thermal expansion coefficient plus the linear fit parameters calculated for all protein MD simulations are given in Tables 1 and 2 for T4 lysozyme and barnase, respectively.

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Table 1. Parameters obtained from the MD simulations of T4 lysozyme mutant proteins a
PDB code 3lzm 172l 1l24 1l30 1l34 1l48 1l69 1l16 1l10 1l15 Protein WT I3C A82P P86L R96H A98V L133A G156D T157I T157V ASA8 ~2 ! 8,143 9,107 8,263 8,135 8,150 8,261 8,042 8,263 8,074 8,214 dASA0dT ~2 {K 1 ! 0.92 6 0.16 0.91 6 0.21 0.57 6 0.23 1.1 6 0.33 1.27 6 0.23 2.0 6 0.45 1.87 6 0.14 1.86 6 0.15 1.47 6 0.15 1.2 6 0.14 a{10 6 ~K 1 ! 113 6 20 100 6 23 69 6 28 136 6 41 157 6 28 242 6 55 223 6 17 225 6 18 182 6 19 146 6 17
exp~app! Tm ~K!

R. Palma and P.M.G. Curmi Table 2. Results obtained for the barnase wild-type protein and a set of mutants a
PDB code 1bnj 1brh 1bsa 1bri 1bsb 1brj 1bsc 1bse 1brk 1bsd Protein WT L14A I51V I76A I76V I88A I88V L89V I96A I96V ASA8 ~2 ! 5,875 5,758 5,750 5,821 5,804 5,711 5,710 5,980 5,855 5,722 dASA0dT ~2 {K 1 ! 0.48 6 0.19 2.1 6 0.28 0.6 6 0.17 0.64 6 0.14 0.75 6 0.13 2.68 6 0.24 0.98 6 0.16 1.34 6 0.19 1.32 6 0.15 0.44 6 0.26 a{10 6 ~K 1 ! 81.7 6 32 360 6 49 105 6 29 110 6 25 129 6 22 469 6 42 173 6 28 224 6 31 224 6 26 77 6 45
exp G25 ~kcal0mol!

337.5 341.0 339.8 336* 330.7 328* 330* 331.6 334.5 335.5*

8.82 4.3 7.7 7.16 7.84 4.8 7.18 8.35 5.67 7.8

a The first column corresponds to the PDB code. The column labeled Protein describes the nature of the mutations. Linear fitting parameters for the results of the MD simulations are presented in the columns labeled ASA8 and ~dASA!0dT. The surface thermal expansion coefficients are exp labeled a. Tm corresponds to melting temperatures at pH 6.5. The error in exp the measurement of Tm was assumed to be 60.5 8K. The asterisk ~*! app marks those approximated values for the melting temperature ~Tm ! found by extrapolation of the melting temperature as function of pH assuming a normal dependence without inversions ~Becktel & Baase, 1987!. The uncertainties in these cases were assumed to be equal to 61.5 K.

a The nature of the mutation is presented in the column labeled Protein. The parameters obtained from the linear fitting of the MD simulation are labeled ASA8 and ~dASA!0dT. The surface thermal expansion coefficients are given in the column labeled a. Experimental values for the exp changes in free energy at 25 8C are given in the column labeled G25 . These values taken from Serrano et al. ~1992! have an uncertainty of 60.1 kcal0mol.

exp at 25 8C, G25 , as the protein is unfolded ~Serrano et al., 1992; exp Table 2!. Figure 3 shows a plot of G25 as a function of our computed as for wild-type and mutant barnases. Again, there is an approximately linear relationship between the computed and experimentally measured stability parameters, with a correlation coefficient of 0.83.

The correlation between surface thermal expansion and stability observed for T4 lysozyme is stronger than that for barnase. There are several possible reasons for this difference. The structures available for T4 lysozyme were complete and hence could be simulated with minimal initial refinement. Thus, the starting structures for T4 lysozyme simulations are very close to the original X-ray structures. For barnase, it was necessary to apply a different strategy. Since various atoms were not present in the Brookhaven Protein Data Bank ~PDB! files of the X-ray structures for different mutant proteins ~Bernstein et al., 1977!, these atoms were rebuilt and the models refined. The initial structure for running the MD simulation was obtained after an unconstrained energy minimization. This

Fig. 2. Surface thermal expansion coefficient a plotted against experimental melting temperature for the T4-lysozyme. a was calculated as ~dASA0 exp dT !0ASA8 for the wild-type and mutant proteins. Tm is the melting temperature obtained from thermal denaturation experiments ~Matthews, exp 1995, 1996!. For Tm , the error estimate was taken as 6 0.5 K. Larger error bars ~6 1.5 K! are drawn for those cases where the melting temperature app was approximated, Tm using the pH dependence of the melting temperature established by Becktel and Baase ~1987! assuming a normal dependence without inversions. Data in the figure are as per Table 1. The linear correlation coefficient for this figure is equal to 0.86.

Fig. 3. Surface thermal expansion coefficient plotted against experimental changes in stability at 25 8C for barnase. The correlation is shown between exp G25 obtained from urea denaturation experiments ~Serrano et al., 1992! and the surface thermal expansion coefficient a calculated as the ratio: ~dASA0dT !0ASA8. Data in the figure are as per Table 2. The linear correlation coefficient for this figure is equal to 0.83.

Correlation between protein expansion and stability resulted in larger deviations between the original X-ray coordinates and the starting structure for the MD simulations ~see Materials and methods!. Another source of difference between T4 lysozyme and barnase is that for the former most mutations are near the protein surface, while for the latter all mutations studied involved core residues. Also, for barnase, the calculated surface thermal expansion coefficient is compared against the changes in the free energy at 25 8C from urea denaturation ~instead of Tm !. However, there is evidence that the stability measurements obtained by different means are comparable ~Privalov, 1979!. Correlation between thermal expansion of model mutant proteins and stability To test the dependence of the method on a prior knowledge of the high resolution structure of the mutant protein, we have investigated the behavior of model mutant protein structures during temperature dependent MD simulations. Twenty-four barnase mutant protein models were generated from the wild-type crystal structure ~Table 3!. These models included most of the mutant proteins of known crystal structure, which were used in the simulations above

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~Table 2!. Quasi-equilibrium simulations were carried out for these models using the same simulation parameters as per the barnase crystal structures. The model mutant proteins showed similar behaviour as the crystal structures ~Table 3; Fig. 4!. exp Figure 4 shows a plot of G25 as a function of our computed as for the model mutant proteins. There is an approximately linear relationship between the computed and experimentally measured stability parameters, with a correlation coefficient of 0.69. There is considerably more spread in the data compared with the simulations based on the crystal structures. However, the same trends are observed and the two plots ~Figs. 3, 4! can be overlayed. Eight of the model simulations correspond to crystal structure based simulations ~L14A, I76A, I76V, I88A, I88V, L89V, I96A, and I96V!. The a-values for the two types of simulation are within error of all but two of the mutant proteins ~I88A and L89V!. These exceptions show abnormally high as calculated from the crystal structure simulations, given their stabilities ~Fig. 3!. Thus, the correlation observed between the thermal stability of mutant proteins and their surface thermal expansion as calculated by MD simulation based on their crystal structures can be mimicked by using computer generated models, albeit with a loss of precision. Importance of the accessible surface area for protein stability

Table 3. Results obtained for the barnase wild-type protein and a set of model mutants a
Protein WT V10A V10T Y13A L14A Y24F I25A I25V Q31S N41D I51A D54A D54N N58D I76A I76V I88A I88V L89V L89T S91A S92A I96A I96V Y103F ASA8 ~2 ! 5,875 5,880 5,782 5,820 5,770 5,877 5,830 5,941 5,731 5,758 5,750 5,705 5,802 5,981 5,833 5,802 5,743 5,720 5,725 5,800 5,872 5,817 5,792 5,888 5,850 dASA0dT ~2 {K 1 ! 0.48 6 0.19 1.58 6 0.17 1.17 6 0.14 0.83 6 0.25 1.80 6 0.20 0.24 6 0.17 0.83 6 0.28 0.42 6 0.18 0.12 6 0.20 1.22 6 0.13 0.83 6 0.15 1.54 6 0.28 0.86 6 0.26 0.40 6 0.37 0.47 6 0.22 0.9 6 0.13 1.43 6 0.21 1.0 6 0.23 0.60 6 0.12 1.29 6 0.16 0.46 6 0.3 1.15 6 0.25 0.80 6 0.30 0.44 6 0.17 1.12 6 0.18 a{10 6 ~K 1 ! 81.7 6 32 270 6 29 202 6 24 142 6 44 311 6 35 41 6 29 142 6 48 71 6 31 21 6 35 213 6 24 145 6 27 270 6 50 148 6 46 67 6 63 80 6 38 157 6 22 249 6 37 176 6 41 106 6 21 223 6 27 79 6 51 197 6 42 140 6 53 70 6 29 192 6 30
exp G25 ~kcal0mol!

8.82 5.35 6.83 5.82 4.3 8.9 5.14 7.99 8.75 6.20 4.10 5.96 7.04 9.13 7.16 7.84 4.8 7.18 8.35 5.54 6.41 5.60 5.67 7.8 8.64

Both the thermal expansion and the denaturation processes involve the proteinsolvent interface, which has been shown to play a central role in protein energetics. In our study, the results come from the analysis of the dynamic changes in protein structure rather than from one static snapshot. In this way, we test all protein interactions and their ability to respond to changes in temperature. Our results show that there is a correlation between the thermal expansion of the protein surface in the folded state during simulation and the changes in protein stability parameters for the folded

a Apart from the wild-type, each starting structure was generated by altering one residue via computer graphics. The nature of the mutation is presented in the column labeled Protein. The parameters obtained from the linear fitting of the MD simulation are labeled ASA8 and ~dASA!0dT. The surface thermal expansion coefficients are given in the column labeled a. Experimental values for the changes in free energy at 25 8C are given in exp the column labeled G25 . These values taken from Serrano et al. ~1992! have an uncertainty of 60.1 kcal0mol.

Fig. 4. Surface thermal expansion coefficient plotted against experimental changes in stability at 25 8C for barnase model mutant proteins. Model mutant proteins were constructed via computer graphics and simulations run as per the mutant barnase crystal structures ~Fig. 3!. The correlation is exp shown between G25 obtained from urea denaturation experiments ~Serrano et al., 1992! and the surface thermal expansion coefficient a for the model mutant proteins calculated as the ratio: ~dASA0dT !0ASA8. Data in the figure are as per Table 3. The linear correlation coefficient for this figure is equal to 0.69. The axes are identical to those in Figure 3 to facilitate comparison of the model to crystal structure results.

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to unfolded state transition. It would appear that the effects of a particular mutation are reflected in changes in surface energy, allowing us to obtain a meaningful measure of stability by studying the folded state alone. The correlation between the surface thermal expansion coefficient and the stability parameters gives one the opportunity to attempt a physical interpretation of the results obtained. Upon heating there are two opposing effects: the ordering effect of all proteinprotein and proteinsolvent interactions and the disordering, denaturing effect of temperature. In the case where the mutations stabilize the structure, proteinprotein and proteinsolvent interactions are in such a balance that they successfully oppose the denaturing effects of temperature. The structure is maintained by an improved set of interactions that also prevent a rapid thermal expansion. Thus, unfolding will occur at higher temperatures. For those mutations that weaken the structure, proteinprotein and proteinsolvent interactions cannot prevent a rapid protein expansion and, similarly, unfolding occurs at lower temperatures.

R. Palma and P.M.G. Curmi to the force field as modified by Engh and Huber ~1991!. A second force field, AMBER-OPLS, was also tested on T4 lysozyme ~data not shown!. The results obtained for the ASA changes using AMBER-OPLS did not satisfy our requirements of smoothness. Probably the reason for this is that this force field should be used with explicit water molecules. Prior to dynamics, strains in the protein models were relieved via energy minimization using the Powell algorithm ~Powell, 1977!. The ASA was determined using the method of Lee and Richards ~1971! as implemented in X-PLOR using a water probe radius of 1.6 . MD simulations were carried out using the Verlet algorithm ~Verlet, 1967! with one femtosecond time steps. During the first 50 ps of MD simulation, the protein was equilibrated at a temperature equal to T1 ~K!. The temperature was maintained by coupling to an external bath using the method of Berendsen et al. ~1984!. After equilibration, the protein was slowly heated from temperature T1 to T2 via the external bath. This was achieved by incrementing the bath temperature by 5 K after every 10 ps of simulation. Trajectory files were saved every 50 fs and subsequently used to calculate the ASA. Slower heating using longer simulations of up to 1.5 ns was also carried out for some mutant proteins ~data not shown!. The resultant ASA vs. temperature plots obtained from these simulations were comparable to those produced by the shorter simulations.

Limitations There are currently several limitations to our result. First, the main difficulty is obtaining simulation conditions where the ASA increases with temperature. Our current strategy involves iteratively varying the coupling parameter and the threshold for selecting the residues for charge neutralization until the wild-type protein surface expands with increasing temperature. The value of the coupling parameter appears to be dependent on the net charge neutralized at the protein surface. Calculations with unconstrained energy minimization of the protein coordinates prior to quasi-equilibrium MD simulation have shown that the behavior of the simulation is sensitive to the starting model. This is linked to the variations in residue solvent exposure after unconstrained minimization compared to constrained minimization ~data not shown!. However, with both procedures, the correlation between the surface thermal expansion coefficient and stability is present. Stronger correlations are observed when the simulations use high resolution X-ray structures as the starting points for MD simulations. Clearly, for a predictive methodology, one requires a model mutant protein structure, based only on the experimental wild-type structure, as a starting point for simulation. Stability parameters calculated from model structures show more scatter than those based on crystal structures. Also, our present data solely regard single-site mutations. Extension to multiple mutations will clearly be important for protein engineering. We are currently developing protocols to address these issues.

Mutant protein structures The proteins T4 lysozyme and barnase were chosen because of the availability of a large number of mutant proteins with both thermodynamic and structural data. The mutant protein structures were obtained from the PDB ~Bernstein et al., 1977!. All structures used in this study were determined by X-ray crystallography with the exception of the computer generated model mutant proteins. The latter were based on the X-ray crystal structure of the wild-type barnase.

T4 lysozyme simulation conditions A switch function was used to truncate the van der Waals interactions over 6 6.5 and a shift function with 7.5 cut-off was used to truncate electrostatic interactions. The dielectric constant was set to unity. Initially, the structures undergo 500 steps of energy minimization, where only the hydrogen atoms are allowed to move. After this period of hydrogen interaction optimization, a further 2,000 steps of constrained energy minimization were carried out to relieve bad nonbonded contacts and other strains in a structure. During this minimization, all atoms were constrained to their original positions by an harmonic potential equal to 20 kcal0mol. This constrained energy minimization produced only small alterations in the structures with an average root-mean-square ~RMS! coordinate difference between the original and final coordinates of less than 0.2 and 0.3 for the backbone and side-chain atoms, respectively. The initial temperature was set equal to 255 K and the final temperature was 395 K. Two hundred structures plus the values for their ASA were obtained for each 5 K temperature step. The total simulation time per T4 lysozyme mutant protein was 310 ps. The coupling parameter fbeta was set equal to 3.50.0066T.

Materials and methods Computer simulations All protein simulations were carried out using the program X-PLOR ~Brnger, 1992!. The computational procedure consisted of the following steps: preparation of the initial structure for each of the mutant proteins investigated, energy minimization of each structure, MD simulation, accessible surface area calculations, and analysis. The structure preparation was done in the explicit hydrogen atom representation. The empirical energy parameters correspond

Correlation between protein expansion and stability For most of the T4 lysozyme mutant proteins, the side chains of the following charged and polar residues were neutralized: R14, K16, K19, T21, K35, N40, K48, R52, N53, N55, K60, K65, R76, R80, T109, R119, K124, R125, K135, R137, N140, N144, R154, K162, and N163. They have an exposed ASA 100 2 . The core residues in most mutant proteins were not coupled to the thermal bath. These were: M6, L7, R8, T26, I27, G28, I29, A42, L46, G56, I58, A63, L66, I78, A98, L99, N101, M102, V111, S117, L121, A129, L133, A146, V149, T152, F153, and Y161. They have an exposed ASA 1.5 2 . Since there are differences between the mutant protein structures, the designation of some of these residues as surface and core varies between mutant proteins. The alterations are often close to the site of mutation. The exceptions are: for the wild-type, I29 is not core; for mutant protein I3C, F4 is core, R8 is not, while D61 is neutralized; for L133A, E129 is no longer core; and for A98V, T152 is no longer core.

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90 has an ASA 5 2 . After including these changes, the final set of values gave an increasing ASA. Model building Model mutant proteins were constructed by replacing the existing residue with the closest rotamer of the new residue. The starting structure was the wild-type barnase structure. All models were made via interactive computer graphics manipulations using the program Torbo-Frodo ~Rousell & Cambillau, 1989!. Data analysis The ASA vs. temperature data were fit to a linear function via a least-squares procedure using Mathematica ~Wolfram, 1991!. This least-squares routine was also used to estimate the correlation between the computed thermal expansion coefficient and the experimental stability parameters. The error estimate in a was calculated from the variance of the gradient of the linear function using standard relationships. The variance of the intercept introduced only small changes, so it was neglected. When fitting ASA vs. temperature data for the mutant proteins, some of the datasets show large perturbations that do not follow the same trend as the rest of the data. In these cases, the linear fit to the data was carried out over a more limited range. However, the merit function was calculated over the whole range so that the error estimate includes the effects of the perturbed regions. The ASA vs. temperature plots for the following three T4 lysozyme mutant proteins were fit over limited ranges. P86L was fit excluding the perturbation at the end of simulation, from 360 to 395 K, R96H excluding the initial perturbation from 255 to 280 K, and A98V excluding the perturbation from 255 to 305 K. Similarly, the ASA vs. temperature plots for three barnase mutant proteins were fit over a limited range. These are: L14A where the fitting was carried out excluding the region from 255 to 310 K, I88A excluding the data from the last 10 K, and I96V excluding the last 20 K. References
Becktel WJ, Baase WA. 1987. Thermal denaturation of bacteriophage T4 lysozyme at neutral pH. Biopolymers 26:619 623. Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR. 1984. Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684 3690. Bernstein FC, Koetzle TF, Williams GJB, Myer EF, Brice MD, Rodgers JR, Kennard O, Shimanoushi T, Tasumi M. 1977. The Protein Data Bank: A computer based archival file for macromolecular structures. J Mol Biol 112:535542. Bond CJ, Wong KB, Clarke J, Fersht AR, Daggett V. 1997. Characterisation of residual structure in the thermally denatured state of barnase by simulation and experiment: Description of the folding pathway. Proc Natl Acad Sci USA 94:1340913413. Brnger AT. 1992. X-PLOR, version 3.1. New Haven, London: Yale University Press. Bull HB, Breese K. 1973. Temperature dependence of partial volume of proteins. Biopolymers 12:23512358. Chothia C. 1974. Hydrophobic bonding and accessible surface area in proteins. Nature 248:338339. Daura X, Jaun B, Seebach D, van Gunsteren WF, Mark AE. 1998. Reversible peptide folding in solution by molecular dynamics simulation. J Mol Biol 280:925932. Eisenberg D, McLachlan AD. 1986. Solvation energy in protein folding and binding. Nature 319:199203. Engh RA, Huber R. 1991. Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallogr A47:392 400.

Barnase simulation conditions The PDB data files for some mutants of barnase have the first two residues plus several other side-chain atoms missing. A decision was taken to work with only 108 residues for all mutant proteins ~i.e., residues 3 to 110!. Due to the missing atoms, a new procedure for minimization was carried out. First, the missing atoms were generated using an interactive graphics program Turbo-Frodo ~Rousell & Cambillau, 1989!. This was followed by a 200 steps unconstrained energy minimization to relieve any bad contacts introduced by the new atoms in the structure, then a constrained harmonic minimization for 2,000 steps and finally an unconstrained minimization for 2,000 steps. At the completion of this procedure, the RMS coordinate differences between the initial and final structures were less than 0.5 for backbone atoms and less than 1.0 for side-chain atoms. Since the barnase mutant proteins that we used involved mutations in the protein core, their protein surfaces are almost identical. Hence, we decided to apply the same simulation conditions to all of them. After the calculation of the ASA for the minimized structures, the side chains of the following charged residues were neutralized: D8, D12, D22, E29, K39, D44, K49, R59, E60, K62, K66, R72, D93, D101, K108, and R110 as they have their exposed ASA 60 2 , which was the threshold chosen for this protein. No friction was applied to residues: V10, A11, L14, P21, N23, A30, W35, G40, L42, A46, S50, I51, G52, L63, 7W1, A74, D75, I76, W78, R87, I88, L89, S91, I96, and T99, which have an exposed ASA 5 2 . A switch function was used to truncate the van der Waals interactions over 7.510 and a shift function with 11 cut-off was used to truncate electrostatic interactions. The dielectric constant was set to four. Initial and final temperatures for MD simulation were 280 and 380 K. The coupling parameter fbeta was set equal to 3.50.0016T. As per T4 lysozyme simulations, the ASA was calculated each 50 fs and the temperature was increased in 5 K steps. The total simulation time was 250 ps giving 5,000 values for ASA. All the mutant proteins under these condition showed a smooth evolution of the ASA, except for L14A. A carefully analysis of the ASAs for the initial structures show that there are a number of differences in this mutant protein as compared with the wild-type. For L14A, the residues N23, W35, A46, W71, and Y78 are exposed 10 2 and hence no longer part of the core, while residue

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Frauenfelder H, Hartmann H, Karplus M, Kuntz ID Jr, Kuriyan J, Parak F, Petsko GA, Ringe D, Tilton RF Jr, Connoly ML, Max N. 1987. Thermal expansion of a protein. Biochemistry 26:254261. Gao J, Kuczera K, Tidor B, Karplus M. 1989. Hidden thermodynamics of mutant proteins: A molecular dynamics analysis. Science 244:10691072. Gilis D, Rooman M. 1996. Stability changes upon mutation of solvent-accessible residues in proteins evaluated by database-derived potentials. J Mol Biol 257:11121126. Gilis D, Rooman M. 1997. Predicting protein stability changes upon mutation using database-derived potentials: Solvent accessibility determines the importance of local versus non-local interactions along the sequence. J Mol Biol 272:276290. Lazaridis T, Karplus M. 1997. New view of protein folding reconciled with the old through multiple unfolding simulations. Science 278:19281931. Lee B, Richards FM. 1971. Interpretation of protein structures: Estimation of static accessibility. J Mol Biol 55:379 400. Li A, Daggett V. 1998. Molecular dynamics simulation of the unfolding of barnase: Characterization of the major intermediate. J Mol Biol 275:677 694. Makhatadze GI, Privalov PL. 1990. Heat capacity of proteins. I. J Mol Biol 213:375384. Matthews BW. 1995. Studies on protein stability with T4 lysozyme. Adv Prot Chem 46:249278. Matthews BW. 1996. Structural and genetic analysis of the folding and function of T4 lysozyme. FASEB J 10:35 41. Ooi T, Oobatake M, Nemethy G, Scheraga HA. 1987. Accessible surface area as a measure of the thermodynamic parameters of hydration of peptides. Proc Natl Acad Sci USA 84:30863090. Powell MJD. 1977. Restart procedures for the conjugate gradient method. Math Programming 12:241254. Privalov PL. 1979. Stability of proteins. Small globular proteins. Adv Protein Chem 33:167241.

R. Palma and P.M.G. Curmi

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