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Energetics of aliphatic deletions in protein cores

Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias & Biocomputation, and Complex Systems Physics Institute (BIFI), Universidad de Zaragoza, 50009-Zaragoza, Spain

(RECEIVED April 7, 2006; FINAL REVISION May 3, 2006; ACCEPTED May 3, 2006)

	Abstract

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
Although core residues can sometimes be replaced by shorter ones without introducing significant changes in protein structure, the energetic consequences are typically large and destabilizing. Many efforts have been devoted to understand and predict changes in stability from analysis of the environment of mutated residues, but the relationships proposed for individual proteins have often failed to describe additional data. We report here 17 apoflavodoxin large-to-small mutations that cause overall protein destabilizations of 0.6–3.9 kcal.mol^–1. By comparing two-state urea and three-state thermal unfolding data, the overall destabilizations observed are partitioned into effects on the N-to-I and on the I-to-U equilibria. In all cases, the equilibrium intermediate exerts a "buffering" effect that reduces the impact of the overall destabilization on the N-to-I equilibrium. The performance of several structure-energetics relationships, proposed to explain the energetics of hydrophobic shortening mutations, has been evaluated by using an apoflavodoxin data set consisting of 14 mutations involving branching-conservative aliphatic side-chain shortenings and a larger data set, including similar mutations implemented in seven model proteins. Our analysis shows that the stability changes observed for any of the different types of mutations (LA, IA, IV, and VA) in either data set are best explained by a combination of differential hydrophobicity and of the calculated volume of the modeled cavity (as previously observed for LA and IA mutations in lysozyme T4). In contrast, sequence conservation within the flavodoxin family, which is a good predictor for charge-reversal stabilizing mutations, does not perform so well for aliphatic shortening ones.

Keywords: protein stability; hydrophobic interaction; protein intermediate; protein folding; protein cavity

	Introduction

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
Globular proteins are organized around well-packed hydrophobic cores that play important roles in protein folding and stability (Chan and Dill 1990; Dill 1990). Tight packing optimizes van der Waals interactions and allows for the minimization of the presence of cavities in proteins. Although small perturbations of core packing arising from mutations are often tolerated by proteins and give rise to small structural readjustments, they may result in large energetic effects (Milla et al. 1994; Munson et al. 1996; Vlassi et al. 1999). Understanding the contribution of buried hydrophobic residues to protein stability is not trivial, and toward that end, considerable experimental work has been carried out to analyze large-to-small side-chain substitutions (Yutani et al. 1987; Matsumura et al. 1988; Kellis et al. 1989; Shortle et al. 1990; Baase et al. 1992; Eriksson et al. 1992b; Serrano et al. 1992; Takano et al. 1997; Xu et al. 1998, 2002; Chen and Stites 2001a). One goal in these studies has been to find out relationships between local protein structural features and the concomitant stability changes observed. For the staphylococcal nuclease, the change in stability upon mutation (G) was related to local packing density (Shortle et al. 1990); for barnase, a linear relationship was proposed between G and an averaged measure of structural perturbations in neighboring atoms (Buckle et al. 1996); for chymotrypsin inhibitor 2 (CI2), G was related to the number of methyl and methylene groups within 6 Å from the mutated residues (Otzen et al. 1995). Subsequent studies have nevertheless shown that the correlations proposed were difficult to generalize to other proteins (Xu et al. 1998). A somewhat simpler approach was proposed in a T4 lysozyme study, where G was approximated by a constant term, related to differences in hydrophobicity between the exchanged residues, plus a term proportional to cavity size (Eriksson et al. 1992b; Xu et al. 1998). As with the other relationships, this one was later shown not to hold in a different protein (ROP [Vlassi et al. 1999]). It thus seems clear that further investigation and assessment are required to determine whether general relationships can be established between local protein characteristics and the G associated with buried mutations.

On the other hand it should be noticed that, as in many other mutational analyses, the contribution of buried hydrophobic residues to protein stability has been mainly studied using two-state proteins, where stability differences can be best determined (Pace and Laurents 1989). While two-state small proteins are very convenient models, they might not be very representative of average proteins, which are larger (i.e., 86% of the human proteins documented in Swiss-Prot [Bairoch and Apweiler 1997] are >15 kDa) and therefore more likely to display equilibrium-folding intermediates. For a three- (or more) state protein, it is useful to distinguish between the free-energy difference of the N-to-I equilibrium, which has been termed relevant stability (Sancho et al. 2002), and the residual stability associated with the I-to-U equilibrium. In previous work on charge-reversal mutations (Campos et al. 2004b), we showed that large overall stabilization of the native state relative to the unfolded one can be obtained using charge-reversal mutations without significantly increasing the relevant stability of the protein. Conversely, it can be anticipated that destabilizations arising from hydrophobic deletions in the core of three-state proteins can be partitioned in various ways, the extremes being the specific destabilization of the native state relative to the intermediate and that of the intermediate relative to the denatured state.

To reassess the contribution of hydrophobic residues to the overall stability of two-state proteins and to investigate how it is partitioned in three-state proteins, we analyze here a set of 17 hydrophobic (Fig. 1; Table 1), large-to-small mutations that have been implemented in the core of the apoflavodoxin from Anabaena PCC7119 (169 residues, 19 kDa) (Maldonado et al. 1998; Sancho 2006). The structure, stability, folding, and cofactor binding of this protein are well characterized (Genzor et al. 1996a,b; Fernandez-Recio et al. 1999; Lostao et al. 2000, 2003; Irun et al. 2001b; Langdon et al. 2001; Campos et al. 2005); the protein has been used to investigate different types of side-chain interactions (Fernandez-Recio et al. 1999; Campos et al. 2005); and it displays the interesting feature of behaving as a two-state protein toward chemical denaturation and as a three-state one toward thermal unfolding (Irun et al. 2001a). Our data indicate that all of the mutations tested induce large overall destabilizations that are unevenly distributed between the N-to-I and I-to-U equilibria in a way that can be fully explained on the basis of the available low-resolution structure of the apoflavodoxin thermal intermediate (Campos et al. 2004a). Moreover, 14 of the 17 apoflavodoxin mutants, together with mutants reported in staphylococcal nuclease, barnase, CI2, ROP, and T4 and human lysozyme, have been jointly analyzed to test the overall performance of the various correlations proposed between structural features and stability changes. According to our analysis, the best predictor of the observed stability changes is cavity volume.

View larger version (59K): [in this window] [in a new window]   Figure 1. C trace of Anabaena apoflavodoxin (PDB code 1FTG) showing, in green, the mutated side-chains.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

View larger version (24K): [in this window] [in a new window]   Figure 2. Circular dichroism spectra of wild type and some of the mutants analyzed (see Campos et al. 2004a for the rest of the mutants) at 25.0° ± 0.1°C. (A) Near-UV, in 50 mM MOPS (pH 7.0). (B) Far-UV, in 5 mM MOPS (pH 7.0), with 15 mM NaCl. Signals of the W66F mutant (and derived) are shown normalized, for the sake of comparison.

View larger version (32K): [in this window] [in a new window]   Figure 3. Urea denaturation curves of wild-type and mutant apoflavodoxins at 25.0° ± 0.1°C (pH 7.0), 50 mM MOPS. The fluorescence signal of the W66F mutant is shown normalized, for the sake of comparison.

One of the earliest and most straightforward structure–energetics relationships proposed to account for the destabilization arising from replacing buried aliphatic residues by shorter ones is that of Kellis et al. (1989). From the analysis of two Ile to Val barnase mutants (I88V, I96V) the protein was found to be destabilized by 1.0–1.6 kcal.mol^–1/methyl group removed. This value was subsequently refined to 1.5 ± 0.6 kcal.mol^–1 (Serrano et al. 1992) by considering two additional barnase mutants (I5V, I76V), one (I72V) from staphylococcal nuclease (Shortle et al. 1990) and one (I47V) from the gene V protein from bacteriophage f1 (Sandberg and Terwilliger 1991). An additional calculation of the G value per –CH₂-group was performed by Pace (1992) adding to the previous set of mutants, four staphylococcal nuclease mutants (I15V, I18V, I92V, and I139V) and a T4 lysozyme (I3V) mutation. A value of 1.3 ± 0.4 kcal.mol^–1 was obtained (Pace 1992). An analogous calculation using only four apoflavodoxin Ile-to-Val mutations (Table 2) gives a value of 2.0 ± 1.0 kcal.mol^–1/methylene group removed, and if all of the 15 Ile-to-Val mutations are jointly considered, the estimation of the contribution to protein stability of a buried methyl/methylene group is set at 1.5 ± 0.8 kcal.mol^–1.

Relevant (N-to-I) and residual (I-to-U) stability of cavity bearing mutants: An energetic "buffering" effect exerted by protein equilibrium intermediates
The thermal unfolding of apoflavodoxin is three-state (Irun et al. 2001a), which allows for investigation of the specific energetics of N-to-I and I-to-U equilibria (Campos et al. 2004a,b, 2005). The thermal unfolding data corresponding to the hydrophobic apoflavodoxin substitutions is summarized in Table 4. All mutations destabilize the two equilibria. For the N-to-I equilibrium, the fall in T_m values ranges from 0.4° to 14.5° and for the I-to-U equilibrium, from 2.2° to 19.9°. As observed in the chemical denaturation, the I109A mutant appears as the most globally (N-to-U) destabilized protein.

View larger version (19K): [in this window] [in a new window]   Figure 4. Comparison of GNU values obtained for wild-type apoflavodoxin and mutants thereof by adding the GNI and GIU terms calculated from global analysis of three-state thermal unfolding curves (Table 3) with those directly measured from two-state urea denaturation (Table 1). The filled circle represents data from mutant I156V, excluded for the correlation due to possible structural relaxations (see text). The dotted line is a unity fit.

This fortunate characteristic, which is likely to be general for proteins displaying equilibrium intermediates, does not come without its negative side. In principle, since proteins are destabilized by hydrophobic cavity-created mutations, they could also be stabilized by the reverse, i.e., by implementing mutations to fill existing cavities. This strategy has been explored in two-state proteins, where it has sometimes led to overall N-to-U stabilization (Ishikawa et al. 1993; Akasako et al. 1997; Lassalle et al. 2001; Ohmura et al. 2001). If the 17 apoflavodoxin mutants are thought of in an inverse manner, as different proteins whose cavities can be filled to yield the largely stabilized wild-type apoflavodoxin, it is clear that most of the stabilization that is achieved (65% on average) goes to increasing the stability of the intermediate against its full unfolding (Table 5; Fig. 5). Only one-third of the stabilization actually increases the relevant stability of the native state against its partial unfolding. In practical terms, this means that the overall stabilization of a protein displaying equilibrium intermediates that can be achieved by cavity filling will probably have, on average, a small impact on the specific stabilization of the N-to-I equilibrium. This effect has already been observed in apoflavodoxin using charge-reversal stabilizing mutations that, in most cases, left the relevant stability unchanged (Campos et al. 2004b). However, when the charge-reversal mutations were introduced in the region of the protein that appears unstructured in the thermal intermediate (Campos et al. 2004), significant increases of the relevant stability were obtained. Similarly, a close look at how the destabilizations associated to aliphatic deletions are partitioned into the two equilibria (Fig. 5) shows that in the two instances where the mutations are introduced in the unstructured region of the intermediate (V139A and L143A) (Campos et al. 2004a), the largest destabilization is also associated to the N-to-I transition. In a recent work (Bueno et al. 2006), several cavities in apoflavodoxin have been filled, and one of the mutants that retain the native conformation is significantly more stable than wild type toward full unfolding. However, as the filling mutation is located in the region where the intermediate displays a native-like conformation, the relevant stability of the mutant is not greater than that of wild type.

View larger version (82K): [in this window] [in a new window]   Figure 5. Partitioning of the global destabilization (N-to-U equilibrium) associated to the mutations implemented into the N-to-I equilibrium, relevant destabilization (gray bar), and the I-to-U equilibrium, residual destabilization (white hatched bar).

View larger version (38K): [in this window] [in a new window]   Figure 6. Apoflavodoxin secondary structure cartoon showing new (continuous thick lines) and previously reported (Campos et al. 2004a) (discontinuous thin lines) -values reporting on the integrity of native side-chain interactions in the equilibrium thermal unfolding intermediate at 317.3 K. The native-like region of the intermediate is formed by the packing of helices 1–5, and strands 1–4 (where the -values are >0.6) and possibly strand 5a. In contrast, the long loop splitting strands 5a and 5b (which contains a small three-stranded b-sheet comprising strands 6, 7, and 8), and two additional loops, are markedly weakened (all -values <0.4) (Campos et al. 2004a). The -values are colored from dark green (native interactions) to red (lost interactions). ID numbers of helices (circles) and strands (rectangles) are marked in light gray, and the positions analyzed in this work are indicated in black.

To evaluate whether the cavities created in apoflavodoxin are likely solvated or not, we have selected two different criteria: first, a volume cutoff, and second, an energetic cutoff. Hubbard et al. (1994) demonstrated that most cavities with volumes >50 ų are hydrated. On the other hand, Takano et al. (2003) showed that buried water molecules stabilize a protein structure. Tight packing of buried water molecules in the interior of proteins provides better van der Waals interactions than those in an empty cavity (Takano et al. 1997), and thus a solvated cavity is more stable than an empty one. The reported average destabilization of Ile/Leu to Ala mutations in a solvated cavity is of 1.5 kcal.mol^–1, much less than the same type of mutation in empty cavities: 3.4 kcal.mol^–1 (Takano et al. 2003). Of all of the apoflavodoxin mutants considered in this work, only mutants L6A, L50A, L105A, I109A, and I156V present cavities >50 ų and could be thus solvated, but all of them show destabilizations >2.5 kcal.mol^–1 (see Table 2). Accordingly, the probability of finding water molecules in the apoflavodoxin mutant cavities is low and they will be considered as empty cavities.

Performance of different structure/energetics relationships for hydrophobic large-to-small mutations in protein cores
The relationship between structural characteristics of protein cores and stability changes arising from large-to-small substitutions is not well understood. Different correlations have been proposed for different data sets corresponding to mutations located in several proteins. Here we reassess, in two ways, the performance of those correlations. First (Fig. 7), we tested them on 14 apoflavodoxin mutants corresponding to single mutations involving aliphatic residues; second (Fig. 8), we tested them on a larger data set comprising mutations from seven different proteins: 14 mutants from apoflavodoxin, two from ROP protein (Vlassi et al. 1999), four from barnase (Serrano et al. 1992), eight from T4 lysozyme (Eriksson et al. 1992a; Xu et al. 1998), three from staphylococcal nuclease (Shortle et al. 1990), three from CI2 (Otzen and Fersht 1995), and two from human lysozyme (Takano et al. 1995, 1997). All of these protein variants are also single mutants involving aliphatic residues completely buried from solvent.

View larger version (22K): [in this window] [in a new window]   Figure 7. Plots of G (wild-type apoflavodoxin free energy of unfolding minus that of mutants) vs. different parameters that describe the environment of the mutated residues. (A) Number of C groups within a sphere of 10 Å radius centered in the C of the mutated residue, (B) differences in side-chain solvent-accessible area buried between wild type and mutant, (C) number of methyl or methylene side-chain groups surrounding (within a sphere of 6 Å radius) the methyl or methylene group deleted upon mutation, (D) change of contacts (calculated as contact parameter nH) relative to the wild-type protein, (E) thermal factors of the mutated residues, as extracted from the PDB file 1FTG, and (F) cavity volume created by the amino acid substitution.

View larger version (25K): [in this window] [in a new window]   Figure 8. Plots of G of unfolding vs. different parameters that describe the surrounding environment of mutated residues in seven different proteins: apoflavodoxin, ROP (Vlassi et al. 1999), barnase (Serrano et al. 1992), T4 lysozyme (Eriksson et al. 1992a; Xu et al. 1998), staphylococcal nuclease (Shortle et al. 1990), CI2 (Otzen and Fersht 1995), and human lysozyme (Takano et al. 1995, 1997). (A) Number of C groups within a sphere of 10 Å radius centered in the C of the mutated residue; (B) differences in side-chain solvent-accessible area buried between wild type and mutant; (C) number of methyl or methylene side-chain groups surrounding (within a sphere of 6 Å radius) the methyl or methylene group deleted upon mutation; (D) change of contacts (calculated as contact parameter nH) relative to the wild-type protein; (E) thermal factors of the mutated residues as extracted from the PDB file of the wild-type protein; (F) cavity volume created by the amino acid substitution.

A further attempt to relate the structure and energetics of hydrophobic large-to-small replacements was carried out on T4 lysozyme mutants for which G was found to correlate (r = 0.96) to the size of the engineered cavities (Eriksson et al. 1992b) with a slope of 22 cal.mol^–1Å^–3 (Xu et al. 1998). This linear correlation was roughly confirmed in other proteins (Takano et al. 1995; Buckle et al. 1996), but more recently it was claimed to be not so general (r = 0.51 for ROP [Vlassi et al. 1999]). However, the correlation performs quite well in the apoflavodoxin data set (r = 0.87) (Fig. 7F) with a slope of 21 cal.mol^–1Å^–3 and an intercept of +1.9 kcal.mol^–1. For the set of seven proteins, the correlation is still reasonable (r = 0.72) (Fig. 8F), with a very similar slope of 22 cal.mol^–1Å^–3 and an intercept of +1.6 kcal.mol^–1.

The work by Matthews’ lab (Eriksson et al. 1992b; Xu et al. 1998) showed that the energetic effect of both Leu or Ile to Ala mutations could be correlated to the volume of the created cavity at a rate of 22 cal.mol^–1Å^–3, and that each type of mutation had, in addition, an intrinsic destabilizing effect that could be attributed to the specific hydrophobicity of the substituted side chain as measured by water/octanol transfer experiments (Fauchere and Pliska 1983). For valine to alanine mutations, the data were more scattered. To investigate whether the same principles can be extended to other proteins and to different mutations, we separated the flavodoxin mutations into three groups and performed specific correlations for Leu to Ala, Val to Ala, and Ile to Val mutations. For the three subsets, the slope of G versus cavity volume plots displays values between 20 and 23 cal.mol^–1Å^–3. The intercepts of the plots, however, differ from 1.4 and 1.5 kcal.mol^–1 for Val to Ala and Leu to Ala mutations to 0.9 kcal.mol^–1 for Ile to Val ones, which is in qualitative agreement with the corresponding water/octanol difference transfer energies of 1.2, 1.9, and 0.7 kcal.mol^–1 (Xu et al. 1998). An equivalent analysis performed in the seven-protein database gives slope values ranging from 20 to 24 cal.mol^–1Å^–3 and intercepts that correlate reasonably well with water-to-octanol transfer energies (r = 0.88) (Fig. 9). The intercepts also correlate well with the ASA of the mutated side chains in the denatured state as calculated using three different types of denatured state models: extended Ala-X-Ala tripeptide (r = 0.79) (Shrake and Rupley 1973), extended Gly-X-Gly tripeptide (r = 0.85) (Miller et al. 1987), and 15-mer in silico modeled peptide (r = 0.82) (Creamer et al. 1995). The data corresponding to the four types of mutations (Ile to Ala, Leu to Ala, Val to Ala, and Ile to Val) investigated in the seven proteins data set were brought to a common, hydrophobicity-independent reference state by substracting the intercepts (Fig. 10). This raises the correlation between G and cavity volume from 0.72 (Fig. 8F) to 0.90. Further attempts to improve the correlation considering additional terms accounting for differences in side-chain entropy or secondary structure propensity did not work. Our analyses thus support the proposal (Eriksson et al. 1992b) that the change in free energy upon replacement of Leu or Ile to Ala can be reasonably explained by a combination of differential hydrophobicity plus a term proportional to cavity volume. In addition, the correlation is now extended to Ile to Val and to Val to Ala mutations. All branching-conservative, side-chain shortenings of purely aliphatic residues are thus explained using the same principles.

View larger version (17K): [in this window] [in a new window]   Figure 9. Correlation between transfer energies and interceptors of linear relationships between cavity volume and experimental G for different types of mutations (Leu to Ala, Val to Ala, Ile to Val, and Ile to Ala) implemented in any of the seven proteins of the data set (see Materials and Methods). The dashed line shows a unity fit.

View larger version (17K): [in this window] [in a new window]   Figure 10. The experimentally determined G values shown in Figure 8F have been brought to a common reference state (hydrophobicity-independent) by subtraction of the intercepts of mutation-type separated plots of GNU vs. volume (see Discussion).

	Conclusion

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
A total of 14 buried, branching-conservative shortening mutations involving apolar aliphatic residues have been implemented in apoflavodoxin, giving rise to large protein destabilization without introducing major changes in protein structure. In the thermal unfolding, the overall N-to-U destabilization is partitioned into destabilization of the native state relative to an unfolding intermediate (Sancho et al. 2002) and destabilization of the intermediate toward its full unfolding, which lowers the destabilizing impact exerted by the mutations on the native conformation. This suggests that stabilization of three- (or more) state proteins by cavity filling may be of limited success, unless the filled cavity is specifically located in an unfolded region of the intermediate.

An analysis of several structure–energetics relationships proposed to explain mutational effects on protein stability associated to apolar-buried shortening mutations was performed using the apoflavodoxin data set and a larger data set comprising aliphatic-shortening mutations described for seven different proteins. The analysis indicates that modeled cavity volume (Machicado et al. 2002) plus differential hydrophobicity (Eriksson et al. 1992b) are good predictors of the observed energetics, as previously proposed (Xu et al. 1998) for a smaller data set of lysozyme T4 mutants of known X-ray structures. In contrast, unlike what has been described for SH3 domains (Di Nardo et al. 2003), sequence conservation analysis seems to not work so well.

	Materials and methods

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
Mutagenesis, protein purification and quantitation, and apoprotein preparation
The Anabaena PCC7119 flavodoxin gene was mutated using the QuikChange site-directed mutagenesis kit (Stratagene). The following substitutions were made: I22V, L50A, L105A, I109A, V160A, W66F, W66F/L44A, and W66F/L105A (Fig. 1), replacing buried hydrophobic residues by smaller ones. In addition, the mutants L6A, V31A, I51V, I52V, A84G, V117A, V139A, L143A, and I156V (Campos et al. 2004) were used in the analysis (Table 1). The 17 mutants were purified as described (Fillat et al. 1991) and, in all cases, the final preparations were homogeneous as judged by SDS-PAGE.

To obtain the apoflavodoxins, the flavin mononucleotide group was removed from the holoproteins by precipitation with trichloroacetic acid (Edmondson and Tollin 1971). Apoflavodoxin concentration was determined from the absorbance at 280 nm using an extinction coefficient of 34.1 mM^–1 cm^–1 (Genzor et al. 1996a), except for W66F and variants containing this mutation, for which an extinction coefficient of 27.6 mM^–1 cm^–1 was used (S. Zorrilla, unpubl.).

Absorbance, fluorescence, and circular dicroism (CD) spectra
Emission fluorescence were recorded at 25.0° ± 0.1°C in 50 mM MOPS (pH 7.0), in an Aminco-Bowman Series 2 fluorimeter. CD spectra were recorded in a Jasco 710 spectropolarimeter at 25.0° ± 0.1°C. Near-UV CD spectra were measured in a 1-cm path-length cuvette with 40 µM apoprotein solutions in 50 mM MOPS (pH 7). Far-UV CD spectra were recorded in a 1-mm path-length cuvette using 20 µM apoprotein solutions in 5 mM MOPS (pH 7), with 15 mM NaCl to obtain the same ionic strength of the 50 mM MOPS buffer. Thermal and urea denaturation curves were recorded in the same instruments.

Urea denaturation equilibrium
Protein samples were prepared by mixing 900-µL urea solutions of different concentrations with 100-µL aliquots of 20 µM apoprotein in 500 mM MOPS (pH 7.0). The unfolding at 25.0° ± 0.1°C was followed, after equilibration for 30 min by measuring a ratio of fluorescence emission (320:360 nm, with excitation at 280 nm; see López-Llano et al. 2006 for details). The urea unfolding of apoflavodoxin at pH 7.0 is two-state (Genzor et al. 1996a). The unfolding curves were analyzed assuming that the free energy of unfolding, G, is a linear function of urea concentration (Pace 1990):

where G_w is the free energy of unfolding in the absence of denaturant, D is the molar concentration of urea, and m is a proportionality constant. The spectroscopic signal of the folded (S_F) and unfolded (S_U) states are assumed to vary linearly with urea concentration (Bolen and Santoro 1988) with slopes m_F and m_U, respectively. Under this assumption, the observed spectroscopic signal follows:

where R is the gas constant and T is the absolute temperature.

Thermal unfolding followed spectroscopically
Four different spectroscopic techniques were used to follow the thermal denaturation of each protein variant. For fluorescence, a ratio of emission (320:360 nm with excitation at 280 nm) was used to minimize the strong temperature dependence of the baselines. Apoprotein concentration was 2 µM in 50 mM MOPS (pH 7). Circular dichroism and absorbance in the near-UV were simultaneously measured at 291 nm using 40 µM apoflavodoxin solution in 50 mM MOPS (pH 7). Circular dichroism in the far-UV (222 nm) was recorded using 1 µM apoprotein solution in 5 mM MOPS (pH 7), with 15 mM NaCl. Near- and far-UV experiments were carried out using a 1-cm path-length cuvette.

Thermal unfolding: Three-state global analysis
Global fitting of the four thermal unfolding curves recorded for each protein (fluorescence, far-UV CD, near-UV CD, and near-UV absorbance) to a three-state model involving native (N), intermediate (I), and unfolded (U) conformations was performed with the program MLAB (Civilized Software, Inc.).

For each protein, the curves were fitted to the following three-state equation, with an adaptation (Campos et al. 2004) of the one previously described for a two-state model (Privalov 1979):

with

where Y is the observed spectroscopic signal; Y_N, Y_I, and Y_D are spectroscopic signals at T = 0 K of the native, intermediate, and denatured states, respectively; m_N, m_I, and m_D are slopes that describe the temperature dependence of the spectroscopic signals of the native, intermediate, and denatured states, respectively; H_NI, T_mNI, and C_pNI correspond, respectively, to the enthalpy change, the melting temperature, and the change in heat capacity of the first transition; and H_IU, T_mIU, and C_pIU to those of the second transition . For each mutant, the four thermal unfolding curves were roughly normalized in order to obtain property value spans of 1–0, which balances the contribution of the different curves in the fitting without having to perform preliminary individual fits. All of the curves were then globally fitted to common H, T_m, and C_p values using MLAB (Civilized Software, Inc.). Due to the typically large errors associated to fitted C_p values, the C_p values were constrained in the fitting to be within the standard deviation interval around the mean of the C_p values calculated without restrictions for 40 different apoflavodoxin mutants as described in Campos et al. (2004a). Standard deviations have been determined as described in Beechem (1992).

Equilibrium –analysis of the thermal intermediate
Inspired in classical -analysis (Fersht et al. 1992) originally devised to map out side-chain interactions in transition states of protein unfolding and in transient intermediates, we have recently developed equilibrium -analysis as a way to investigate the integrity in equilibrium intermediates of side-chain interactions present in native states (Irun et al. 2001a; Campos et al. 2004; Korkin et al. 2005). In this analysis, values of 1 or 0 represent native interactions that are present or absent, respectively, in the intermediate, while intermediate values, especially those at internal positions, are interpreted as indicative of weakened interactions. Equilibrium -values are calculated as:

Although Equations 4 and 5 provide a way to calculate G_NI and G_IU values, the inaccuracy of the fitted C_p values misadvises it, and we use a simplified equation that allows us to calculate equilibrium values at a precise reference temperature (chosen at 317.3 K). The details of the fitting have been previously described (Campos et al. 2004).

Modeling of protein mutant structure: Cavity volume and solvent accessibility calculations
The modeling of the mutant structures was done as described in Machicado et al. (2002). Energy minimizations were carried out using the CHARMm force field (Brooks et al. 1983). A cutoff distance of 11 Å was used for nonbonded interactions (with a smoothing from 8 Å). Each structure was energy-minimized, without restrictions, using the method of steepest descent (2000 steps). Minimizations were started from the X-ray structure of wild-type Anabaena PCC 7119 apoflavodoxin after having implemented the appropriate in silico mutation. Cavity volume was calculated with a probe radius of 1.4 Å using the Connolly method (Connolly 1983) as implemented in SwissPDBViewer (Guex and Peitsch 1996).

The difference in solvent-accessible area buried in wild-type and mutant folded proteins was estimated as described (Serrano et al. 1992). First, the solvent accessibility of the residue of interest in the wild-type structure (calculated using the Lee and Richards algorithm [Lee and Richards 1971]) was subtracted from that calculated for that residue in an extended tripeptide Gly-X-Gly (Miller et al. 1987) to give a value A. In parallel, the in silico mutation was performed by deleting the appropriate carbon atoms from the coordinate file of the wild-type protein, and the solvent-accessible area of the mutated residue in the protein was calculated. This value was subtracted from the one calculated in the tripeptide to give a value B. The difference between the two, A–B, is the loss of solvent-accessible area buried in the folded protein upon mutation.

Cavity volumes and differences in solvent-accessible area were also calculated in mutants from ROP (Vlassi et al. 1999), barnase (Serrano et al. 1992), T4 lysozyme (Eriksson et al. 1992a; Xu et al. 1998), staphylococcal nuclease (Shortle et al. 1990), CI2 (Otzen and Fersht 1995), and human lysozyme (Takano et al. 1995, 1997).

Analysis of packing density
The number of contacts between residues located at the site of mutation was calculated on the wild-type structure (PDB code 1ftg [PDB] ) and modeled mutants using the WHATIF software package (Vriend 1990). Similar calculations were made in mutant proteins from ROP (1rop), barnase (1bni), T4 lysozyme (1l63), staphylococcal nuclease (1stn), chymotrypsin inhibitor (2ci2), and human lysozyme (1lz1).

Residue contact parameters, n_H, were calculated as defined (Vlassi et al. 1999). Each atom i of a given core residue h (site of mutation) is assumed to make N_i contacts to j atoms within a 6 Å radius. Each contact is weighted by 1/d_ij, the inverse of the distance between atom i and j, and by 1/r_acc,j, the inverse of the relative accessibility area (calculated using NACCESS [Hubbard et al. 1991]) of the residue to which atom j belongs.

Packing densities were calculated in two ways: as the number of methyl and methylene groups within 6 Å of any side-chain atom removed by mutation (Otzen and Fersht 1995) and as the number of C carbons within a sphere of 10 Å radius from the C of the mutated residue (Shortle et al. 1990). These calculations were performed using the MMTK software (Hinsen 2000).

Flavodoxin sequence alignment
A total of 75 flavodoxins homologs of the Anabaena PCC7119 flavodoxin sequence were identified with BLAST (Altschul et al. 1997) and preliminarily aligned with CLUSTALW (Thompson et al. 1994). Incomplete sequences were discarded and the remaining sequences realigned. Each sequence was weighted following an established procedure (Henikoff and Henikoff 1994) so that weighted percentages of conservation of residues at each flavodoxin position mutated in this work were calculated.

	Footnotes

 
Reprint requests to: Javier Sancho, Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, E 50009-Zaragoza, Spain; e-mail: jsancho@unizar.es; fax: +34-976-76-21-23.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062274906.

	Acknowledgments

 
We acknowledge financial support from grant BFU2004-01411 (Spain). M.B., L.A.C., and J.E. were supported by MEC fellowships.

	References

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
Adamek D.H., Guerrero L., Blaber M., Caspar D.L. 2005. Structural and energetic consequences of mutations in a solvated hydrophobic cavity. J. Mol. Biol. 346: 307–318.[CrossRef][Medline]

Akasako A., Haruki M., Oobatake M., Kanaya S. 1997. Conformational stabilities of Escherichia coli RNase HI variants with a series of amino acid substitutions at a cavity within the hydrophobic core. J. Biol. Chem. 272: 18686–18693.[Abstract/Free Full Text]

Alber T., Sun D.P., Nye J.A., Muchmore D.C., Matthews B.W. 1987. Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein. Biochemistry 26: 3754–3758.[CrossRef][Medline]

Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402.[Abstract/Free Full Text]

Baase W.A., Eriksson A.E., Zhang X.J., Heinz D.W., Sauer U., Blaber M., Baldwin E.P., Wozniak J.A., Matthews B.W. 1992. Dissection of protein structure and folding by directed mutagenesis. Faraday Discuss. 93: 173–181.

Bairoch A. and Apweiler R. 1997. The SWISS-PROT protein sequence database: Its relevance to human molecular medical research. J. Mol. Med. 75: 312–316.[CrossRef][Medline]

Beechem J.M. 1992. Global analysis of biochemical and biophysical data. Methods Enzymol. 210: 37–54.[Medline]

Bolen D.W. and Santoro M.M. 1988. Unfolding free energy changes determined by the linear extrapolation method. 2. Incorporation of G^N-U values in a thermodynamic cycle. Biochemistry 27: 8069–8074.[CrossRef][Medline]

Brooks B., Bruccoleri R., Olafson B., States D., Swaminathan S., Karplus M. 1983. CHARMM: A program for macromolecular energy, minimisation, and dynamics calculations. J. Comput. Chem. 4: 187–217.

Buckle A.M., Henrick K., Fersht A.R. 1993. Crystal structural analysis of mutations in the hydrophobic cores of barnase. J. Mol. Biol. 234: 847–860.[CrossRef][Medline]

Buckle A.M., Cramer P., Fersht A.R. 1996. Structural and energetic responses to cavity-creating mutations in hydrophobic cores: Observation of a buried water molecule and the hydrophilic nature of such hydrophobic cavities. Biochemistry 35: 4298–4305.[CrossRef][Medline]

Bueno M., Cremades N., Neira J.L., Sancho J. 2006. Filling small, empty protein cavities: Structural and energetic consequences. J. Mol. Biol. 358: 701–712.[CrossRef][Medline]

Campos L.A., Bueno M., Lopez-Llano J., Jiménez M.A., Sancho J. 2004a. Structure of stable protein folding intermediates by equilibrium -analysis: The apoflavodoxin thermal intermediate. J. Mol. Biol. 344: 239–255.[CrossRef][Medline]

Campos L.A., Garcia-Mira M.M., Godoy-Ruiz R., Sanchez-Ruiz J.M., Sancho J. 2004b. Do proteins always benefit from a stability increase? Relevant and residual stabilisation in a three-state protein by charge optimisation. J. Mol. Biol. 344: 223–237.[CrossRef][Medline]

Campos L.A., Cuesta-Lopez S., López-Llano J., Falo F., Sancho J. 2005. A double-deletion method to quantifying incremental binding energies in proteins from experiment. Example of a destabilizing hydrogen bonding pair. Biophys. J. 88: 1311–1321.[Abstract/Free Full Text]

Chan H.S. and Dill K.A. 1990. Origins of structure in globular proteins. Proc. Natl. Acad. Sci. 87: 6388–6392.[Abstract/Free Full Text]

Chen J. and Stites W.E. 2001a. Energetics of side chain packing in staphylococcal nuclease assessed by systematic double mutant cycles. Biochemistry 40: 14004–14011.[CrossRef][Medline]

Chen J. and Stites W.E. 2001b. Packing is a key selection factor in the evolution of protein hydrophobic cores. Biochemistry 40: 15280–15289.[CrossRef][Medline]

Connolly M.L. 1983. Solvent-accessible surfaces of proteins and nucleic acids. Science 221: 709–713.[Abstract/Free Full Text]

Creamer T.P., Srinivasan R., Rose G.D. 1995. Modeling unfolded states of peptides and proteins. Biochemistry 34: 16245–16250.[CrossRef][Medline]

Dill K.A. 1990. Dominant forces in protein folding. Biochemistry 29: 7133–7155.[CrossRef][Medline]

Di Nardo A.A., Larson S.M., Davidson A.R. 2003. The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core. J. Mol. Biol. 333: 641–655.[CrossRef][Medline]

Edmondson D.E. and Tollin G. 1971. Flavin-protein interactions and the redox properties of the Shethna flavoprotein. Biochemistry 10: 133–145.[CrossRef][Medline]

Eriksson A.E., Baase W.A., Wozniak J.A., Matthews B.W. 1992a. A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene. Nature 355: 371–373.[CrossRef][Medline]

Eriksson A.E., Baase W.A., Zhang X.J., Heinz D.W., Blaber M., Baldwin E.P., Matthews B.W. 1992b. Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. Science 255: 178–183.[Abstract/Free Full Text]

Eyal E., Najmanovich R., Edelman M., Sobolev V. 2003. Protein side-chain rearrangement in regions of point mutations. Proteins 50: 272–282.[CrossRef][Medline]

Fauchere J.L. and Pliska V. 1983. Hydrophobic parameters of amino-acid side-chains from the partitioning of N-Acetyl-amino-acid amides. Eur. J. Med. Chem. 18: 369–375.

Fernandez-Recio J. and Sancho J. 1998. Intrahelical side chain interactions in -helices: Poor correlation between energetics and frequency. FEBS Lett. 429: 99–103.

Fernandez-Recio J., Romero A., Sancho J. 1999. Energetics of a hydrogen bond (charged and neutral) and of a cation- interaction in apoflavodoxin. J. Mol. Biol. 290: 319–330.[CrossRef][Medline]

Fersht A.R., Matouschek A., Serrano L. 1992. The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224: 771–782.[CrossRef][Medline]