Stabilization of apoflavodoxin by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln

María Pilar Irún, Susana Maldonado and Javier Sancho1,

Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Knowledge of protein stability principles provides a means to increase protein stability in a rational way. Here we explore the feasibility of stabilizing proteins by replacing solvent-exposed hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln. The rationale behind this is a previous observation that, in some cases, neutral hydrogen bonds may be more stable that charged ones. We identified, in the apoflavodoxin from Anabaena PCC 7119, three surface-exposed aspartate or glutamate residues involved in hydrogen bonding with a single partner and we mutated them to asparagine or glutamine, respectively. The effect of the mutations on apoflavodoxin stability was measured by both urea and temperature denaturation. We observed that the three mutant proteins are more stable than wild-type (on average 0.43 kcal/mol from urea denaturation and 2.8°C from a two-state analysis of fluorescence thermal unfolding data). At high ionic strength, where potential electrostatic repulsions in the acidic apoflavodoxin should be masked, the three mutants are similarly more stable (on average 0.46 kcal/mol). To rule out further that the stabilization observed is due to removal of electrostatic repulsions in apoflavodoxin upon mutation, we analysed three control mutants and showed that, when the charged residue mutated to a neutral one is not hydrogen bonded, there is no general stabilizing effect. Replacing hydrogen-bonded charged Asp or Glu residues by Asn or Gln, respectively, could be a straightforward strategy to increase protein stability.

Keywords: charged residues/conformational stability/flavodoxin/hydrogen bond/thermostability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are many instances where increasing the conformational stability of a protein at room temperature or increasing its thermostability may be required. Different rational approaches have been exploited to that end that, in some cases, have proved useful (Nicholson et al., 1988Go; Matsumura et al., 1989; Shih and Kirsch, 1995; Zhang, et al., 1995Go; Villegas et al., 1996Go; Grimsley et al., 1999Go; Williams et al., 1999Go). As the knowledge on the interactions influencing protein stability increases, new strategies may be devised that, ideally, should be general and easy to implement.

We studied protein stability and folding principles using the apoflavodoxin from Anabaena PCC 7119 as a model protein (Maldonado et al., 1998bGo). The structure and energetics of the wild-type form have been described (Genzor et al., 1996aGo, bGo; Maldonado et al., 1998aGo). Its urea unfolding follows a two-state mechanism (Genzor et al., 1996bGo). We have recently focused on the role of hydrogen bonds in protein stability. Despite the fact that these interactions were very early identified as playing a most important role in shaping protein structure (Pauling et al., 1951Go), their contribution to protein stability has been and is still widely debated (Fersht, 1987Go; Honig and Yang, 1995Go; Lazaridis et al., 1995Go; Sippl et al., 1996Go; Myers and Pace, 1996Go). Using apoflavodoxin, we quantified the strength of a side-chain/side-chain hydrogen bond involving a histidine residue (Fernández-Recio et al., 1999Go) and we found, unexpectedly, that the hydrogen bond is stronger when the histidine is neutral. If this were applicable to other hydrogen bonds, a simple strategy towards protein stabilization would involve engineering mutations to transform charged hydrogen bonds into neutral ones. This can be best done by replacing aspartate or glutamate residues by their corresponding neutral isosteric amides, that retain hydrogen-bonding capabilities. To test the feasibility of this strategy, we selected, in apoflavodoxin, three surface-exposed hydrogen bonds involving acidic residues and prepared the appropriate mutants. In all cases, the mutant proteins are more stable at both 25°C and higher temperatures. Additional mutational and buffer- condition control experiments ruled out that the stabilization observed is due to removal of electrostatic repulsions upon mutation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Choice of mutations

Three mutations were designed (E16Q, D96N and D100N) to replace aspartic and glutamic residues involved in surface exposed hydrogen bonds (E16–Y8, D96–N128 and D100–N97) by their isosteric neutral equivalents, asparagine or glutamine (Figure 1Go). Three additional control mutations were designed (E61A, D75A, D126A) where surface-exposed but not hydrogen-bonded aspartic or glutamic residues are replaced by alanine. For these control mutants, mutation to alanine was preferred because, although the charged mutated residues are not hydrogen bonded in the wild-type structure, a replacement by their corresponding amides could have resulted in new interactions occurring with previously unsuitable hydrogen bond acceptors in the neighbourhood, since the amides can act as donors.




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Fig. 1. Wild-type apoflavodoxin ribbon diagram showing the acidic residues mutated and their hydrogen bond partners, when applicable. (a) Residues involved in hydrogen bonds [E16, D96 and D100 (bonded to Y8, N128 and N97, respectively)]; (b) control residues (E61, D75, D126).

 
Mutagenesis

All Anabaena PCC 7119 flavodoxin mutants were prepared by the method of Deng and Nickoloff (1992) directly on the expression plasmid pTrc 99a (Fillat et al., 1991Go). The wild-type protein was mutated to D126A and a wild-type variant carrying the mutation W120F (pseudo-wild-type) was mutated to D100N, E16Q, D96N, E61A and D75A. The pseudo-wild-type form (which is sometimes used in our laboratory because it shows certain advantageous kinetic properties not related to this work) displays a structure and stability behaviour very similar to those of wild-type (not shown). The stability of each mutant apoflavodoxin was compared with that of its appropriate reference wild-type protein. Double-stranded DNA was isolated from the plasmid and the entire flavodoxin gene was sequenced to detect the mutations and to ensure that no additional mutations occurred elsewhere.

Protein expression and purification

Flavodoxin mutants were purified by an adaptation of the method described by Fillat et al. (1991). A 100 ml culture of Escherichia coli TG1 cells was grown overnight in Luria broth medium supplemented with ampicillin (50 mg/ml). Thirteen 1 l bottles containing 300 ml of the same medium with ampicillin were inoculated with 3 ml of culture and cells were grown at 37°C, pH 7, 150 r.p.m. until the optical density of the culture at 600 nm reached 1.2. At this point, isopropyl-ß-thiogalactopyranoside was added at a 1 mM final concentration. Cultures were grown overnight at 150 r.p.m., 37°C. After cooling, cells were harvested by centrifugation in a JA-14 rotor from Beckman. The cell-paste (7–8 g) was washed into 0.15 M NaCl and frozen. The cell-paste was resuspended in 70 ml of 50 mM Tris–HCl, pH 8 (containing 1 mM EDTA, 1 mM mercaptoethanol and 10 µM phenylmethyl sulphonyl fluoride). The suspension was placed in an ice-bath and 50 mg of flavin mononucleotide were added before it was sonicated (6x45 s with a 30 s interval between each treatment). The extract was centrifuged for 1 h at 13 000 r.p.m. in a JA-14 Beckman rotor, the supernatant was brought to 65% ammonium sulphate saturation, centrifuged as above to remove unwanted proteins and loaded on to a DE-52 column equilibrated in 65% saturated ammonium sulphate in Tris buffer. The column was washed with the same buffer until most of the excess flavin mononucleotide was eluted. Flavodoxin was then eluted with 2 l of a linear ammonium sulphate gradient (from 65 to 0%) in Tris buffer. Fractions with OD464/OD280 ratios of >=0.13 were pooled, dialysed against 3x5 l of 5 mM Tris–HCl, pH 8 and loaded on to a second DE-52 column equilibrated in the same buffer. The protein was eluted with 2 l of a linear NaCl gradient (from 0 to 0.5 M). Fractions with OD464/OD280 >=0.16 were pooled. All preparations were homogeneous by SDS–PAGE.

Apoflavodoxin preparation

The flavin mononucleotide group was removed from the holoprotein by treatment with trichloroacetic acid (Edmondson and Tollin, 1971Go).

Protein concentration determination

The concentrations of mutant and wild-type apoflavodoxins were determined from their absorbances at 280 nm using an extinction coefficient of 34.1 mM–1 cm–1 for the wild-type protein (Genzor et al., 1996bGo) and 27.7 mM–1 cm–1 for the W120F mutants (unpublished results).

Absorbance, fluorescence and circular dichroism (CD) measurements

Emission fluorescence and CD spectra were recorded in a Kontron SMF 25 fluorimeter and in a Jasco 710 spectropolarimeter, respectively, at 25.0 ± 0.1°C in 50 mM MOPS, pH 7.0. The thermal and urea denaturation curves were recorded in the same instruments.

Equilibrium urea denaturation

Protein samples were prepared by mixing urea solutions (900 µl) of different concentrations with 100 µl aliquots of 20 µM apoprotein in 500 mM MOPS, pH 7.0. When stated, the urea solution contained choline chloride (0.5 M after mixing with the protein aliquot). The unfolding at 25.0 ± 0.1°C was followed, after equilibration for 45 min, by measuring the emission fluorescence at 320 nm (excitation at 280 nm). The urea unfolding of apoflavodoxin at pH 7.0 and low ionic strength (I = 0.019 M; 50 mM MOPS) is two-state (Genzor et al., 1996bGo). We show here that the two-state behaviour also holds at higher ionic strength when either urea or guanidinium hydrochloride is used as denaturant (see Results). Accordingly, the urea unfolding data were analysed assuming a two-state model (Pace et al., 1989Go). The free energy of unfolding, {Delta}G, is considered to be a linear function of urea concentration according to

(1)
where {Delta}Gw is the free energy of unfolding in water, D is the molar concentration of urea and m is a proportionality constant. The spectroscopic signals of the folded (SF) and unfolded states (SU) are assumed to vary linearly with urea concentration (Santoro and Bolen, 1988Go), with slopes mF and mU, respectively. Under these assumptions, the observed spectroscopic signal follows the equation

(2)
where R is the gas constant and T the absolute temperature.

Since the far- and near-UV CD spectra of the proteins (Figure 2Go) and their fluorescence spectra (not shown), indicate that the proteins are very similar spectroscopically, great care was taken during the analysis that similar slopes for the urea dependency of the native and unfolded signals (mF and mU) were obtained. This contributes to reducing the well-known greater errors in m values (as compared with half urea values) that are so frequently found in urea unfolding data analysis (Pace, 1990Go; Serrano et al., 1992Go). This, in turn, increases the reliability of a direct comparison of {Delta}Gw values of different protein variants. Nevertheless, the difference in protein stability among protein variants is best calculated from the average m value of the different proteins together with the difference in half urea (provided the differences in m values among the protein variants are not large or systematic; see Serrano et al., 1992, for a discussion).



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Fig. 2. Circular dichroism spectra of pseudo-wild-type and mutants involved in hydrogen bonds. (a) Near-UV circular dichroism in 50 mM MOPS, pH 7.0, at 25.0 ± 0.1°C; (b) far-UV circular dichroism in 5 mM MOPS, 15 mM NaCl, pH 7.0, at 25.0 ± 0.1°C.

 
Thermal denaturation followed by fluorescence: two-state analysis

Thermal unfolding curves were acquired using fluorescence emission. To minimize the strong temperature dependence of fluorescence, the ratio of emission at two different wavelengths, 320 and 360 nm (excitation at 280 nm) was used. The buffer was 50 mM MOPS, pH 7.0 and the protein concentration was 2 µM.

Thermal denaturation data were fitted to the equation for a two-state equilibrium:

(3)
where {Delta}G(T) follows the equation

(4)
where {Delta}Cp is the unfolding heat capacity difference and {Delta}H(Tm) is the enthalpy at the melting temperature (Pace et al., 1989Go). Fitting temperature-unfolding data to Equation 3Go allows an accurate determination of Tm and a reasonable determination of {Delta}H(Tm). The {Delta}Cp values, however, are less reliable (Pace et al., 1989Go) and they will not be reported.

Thermal unfolding followed by four different spectroscopic techniques: three-state global analysis

In addition to the fluorescence monitored unfolding curves, thermal unfolding curves of all apoflavodoxin variants were monitored by circular dichroism and absorbance measurements in the near-UV region (simultaneously measured at 291 nm in a Jasco J-710 spectropolarimeter), using 35–40 µM apoflavodoxin in 50 mM MOPS, pH 7.0. Further thermal unfolding curves were similarly measured by circular dichroism in the far-UV region (222 nm), using 1 µM apoprotein in 5 mM MOPS, pH 7.0 (plus NaCl to keep the buffer ionic strength constant).

Global fitting of the four thermal unfolding curves (fluorescence, far-UV CD, near-UV CD and near-UV absorbance) of each protein variant to a three-state model involving native (N), intermediate (I) and unfolded (U) conformations was performed with the program MLAB (from Civilized Software). The approach was that of Luo et al. (1995). Each thermal denaturation curve was preliminarily fitted to the equation for a two-state equilibrium (Equation 3Go). The data were then converted to apparent fractions of unfolded protein, Fapp, using Equation 5Go:

(5)
where Y is the observed signal and YU and YN are the optical signals for unfolded and native protein, respectively. Fapp is related to the fractional populations of the intermediate and unfolded states (FI and FU) by the equation

(6)
where Z is a constant that describes the spectroscopic resemblance of the I state to the U state. From the relationship between fractional populations and equilibrium constants, Fapp can be calculated using Equation 7Go:

(7)
where

(8)

(9)
In each fitting, all thermodynamic parameters ({Delta}H, {Delta}Cp and Tm) were globally constrained, while the Z values were allowed to vary among the different spectroscopic techniques.


    Results
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Spectral characterization of mutant structural integrity

In the absence of X-ray structures, spectroscopic techniques provide means to investigate whether mutations performed in a protein have caused marked perturbations in the overall structure. We report in Figure 2Go the near- and far-UV CD spectra of pseudo-wild-type and the three mutant proteins where charged surface-exposed residues have been mutated to the neutral corresponding amides. As the figure shows, the spectra of the mutants cannot be distinguished from those of the pseudo-wild-type protein. This indicates that the percentage of secondary structure and the tertiary interactions responsible for the near-UV CD spectra are the same in the four proteins. The same applies to the three control mutants and their corresponding wild-type proteins (spectra not shown). Additionally, the emission fluorescence spectrum of each mutant protein is identical with that of the wild-type reference (not shown), indicating that the degree of tryptophan solvent exposure is the same.

Two-state unfolding of apoflavodoxin

The two-state urea unfolding behaviour of the apoflavodoxin from Anabaena in 50 mM MOPS, pH 7 (I = 0.019 M) has been reported (Genzor et al., 1996bGo) from the perfect superposition of the fluorescence and far-UV CD unfolding curves. We extended the analysis to higher ionic strengths and to guanidinium hydrochloride unfolding. Figure 3Go shows the fluorescence and far-UV CD apoflavodoxin unfolding curves in a pH 7 phosphate buffer of ionic strength 0.18 M. As shown by the data (raw data in upper panel and fraction folded in lower panel), the superposition of the curves is very good for both the urea and the guanidinium chloride experiments. The two-state behaviour of apoflavodoxin in the low ionic strength MOPS buffer (Genzor et al., 1996bGo) is thus retained at this higher ionic strength. We assume in our analysis that the presence of choline chloride in some of the experiments does not change this behaviour.



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Fig. 3. Chemical denaturation of wild-type apoflavodoxin at pH 7.0, 25.0 ± 0.1°C and an ionic strength of 0.18 M (100 mM sodium phosphate). Circles, guanidinium hydrochloride denaturation; squares, urea denaturation; open symbols, circular dichroism at 222 nm; closed symbols, fluorescence. (a) Raw data; (b) fraction folded.

 
Conformational stability at 25°C, at low ionic strength

The stability consequences of replacing negatively charged hydrogen-bonded residues by neutral isosteric residues were investigated by urea denaturation equilibrium unfolding. Three such charged residues were individually mutated to either asparagine or glutamine (Table IGo) in order to remove the charge associated with the hydrogen bond while retaining the hydrogen bonding capability. Each mutated residue is involved in just one hydrogen bond.


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Table I. Hydrogen bonds mutated
 
The three mutant apoflavodoxins (E16Q, D96N and D100N) can be unfolded by moderate urea concentrations (Figure 4aGo) and the fluorescence unfolding curves can be fitted to two-state transitions using Equation 2Go. The slope of the observed transitions (m in Equation 1Go) is similar for all these proteins (with a spread of ±4%, Table IIGo), which suggests that no major structural rearrangements occurred upon mutation. A direct comparison of the calculated {Delta}G values in water indicates the mutants are more stable than pseudo-wild-type by 0.32–0.52 kcal/mol. Comparison of the differences in the more precisely determined urea concentrations of mid-denaturation shows that the mutants display higher values than pseudo-wild-type by 0.14–0.26 M (Table IIGo). This indicates that the mutations stabilize the protein from 0.29 to 0.54 kcal/mol. The average stabilizing effect is 0.43 kcal/mol.



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Fig. 4. Urea unfolding curves of wild-type and mutant apoflavodoxins in 50 mM MOPS, pH 7.0, at 25.0 ± 0.1°C, followed by fluorescence emission at 320 nm (excitation at 280 nm). The raw data are represented. Solid lines are fits to the two-state Santoro–Bolen equation. (a) Pseudo wild-type and mutants involved in hydrogen bonds; (b) wild-type, pseudo-wild-type and mutants not involved in hydrogen bonds.

 

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Table II. Stability of wild-type and mutant apoflavodoxins at low ionic strength as measured by urea denaturationa
 
Three additional control mutants were designed so that surface-exposed, but not protein hydrogen-bonded, aspartate or glutamate residues were replaced by alanines. The mutant stabilities were measured and analysed in the same way (Figure 4bGo) and the results are shown in Table IIGo. Two of the three mutations (E61A and D126A) did not significantly stabilize or destabilize the protein, their stability being very close to that of the corresponding wild-type. This rules out the possibility that the folded structure of apoflavodoxin can be generally stabilized by simply removing negatively charged groups. One of the mutants (D75A), nevertheless, did show a stabilizing effect, which prompted additional stability measurements at high ionic strength.

Conformational stability at 25°C, at high ionic strength

In order to minimize electrostatic effects associated with the removal of charges from the protein, the influence of the six mutations on the stability of apoflavodoxin was also measured in the presence of 0.5 M choline chloride. The choline salt was used to avoid cation binding to the protein, which seems to take place when smaller cations are used (unpublished results).

The results at this higher ionic strength (Figure 5Go, Table IIIGo) mimic closely those obtained at low ionic strength, which indicates that the stabilizations observed at low ionic strength are not due to relief of electrostatic repulsions. For the three hydrogen-bonded mutants, the stabilizations observed range from 0.35 to 0.60 (directly calculated from the difference in the determined {Delta}G values in water) or from 0.29 to 0.57 kcal/mol (average 0.46 kcal/mol) when determined from the differences in the urea concentrations of mid- denaturation. As for the control mutants, two of them show hardly any stabilization (as at low ionic strength) and the one that was more stable than wild-type at low ionic strength (0.52 kcal/mol) is similarly more stable at high ionic strength (0.46 kcal/mol).



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Fig. 5. Urea unfolding curves of wild-type and mutant apoflavodoxins in 50 mM MOPS, 0.5 M choline chloride, pH 7.0, at 25.0 ± 0.1°C, followed by fluorescence emission at 320 nm (excitation at 280 nm). The raw data are represented. Solid lines are fits to the two-state Santoro–Bolen equation. (a) Pseudo wild-type and mutants involved in hydrogen bonds; (b) wild-type, pseudo-wild-type and mutants not involved in hydrogen bonds.

 

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Table III. Stability of wild-type and mutant apoflavodoxins at high ionic strength as measured by urea denaturationa
 
Fluorescence thermal unfolding with a two-state analysis

The effect of the mutations on the thermostability of apoflavodoxin was first determined from thermal denaturation experiments using a ratio of fluorescence emission intensities at two different wavelengths to minimize non-structure-related fluorescence changes (Figure 6Go). Simple sigmoidal unfolding curves were obtained that were fitted to Equation 3Go (two-state model). When this equation is used {Delta}H(Tm), {Delta}Cp and Tm values are obtained, but the error in {Delta}Cp is usually large and only Tm and {Delta}H(Tm) are reported in Table IVGo. The transitions observed are reversible (>90%) and they do not depend on protein concentration from 0.5 to 5 µM (not shown). In line with what was observed in the urea denaturation experiments, the three mutations in which a hydrogen bond forming a negatively charged group has been replaced by a neutral isosteric one display higher temperatures of mid-denaturation (from 2.1 to 3.2°C higher; average 2.8°C). In contrast, two of the mutations of non-hydrogen-bonded charged groups do not show any stability effect above the error level. The D75A mutant is nevertheless more stable than its wild-type, as found by urea denaturation.



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Fig. 6. Two-state analysis of the thermal denaturation of apoflavodoxin followed by fluorescence. All samples were in 50 mM MOPS, pH 7.0 and the protein concentration was 2 µM. (a) Pseudo-wild-type and mutants involved in hydrogen bonds; (b) wild-type, pseudo-wild-type and mutants not involved in hydrogen bonds.

 

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Table IV. Thermal denaturation of apoflavodoxin followed by fluorescence: two-state analysisa
 
Three-state global analysis of fluorescence, far-UV CD, near-UV CD and absorbance thermal unfolding curves

We notice that the two-state fits of the fluorescence thermal unfolding curves (Figure 6Go) are an oversimplification of the real three-state thermal unfolding mechanism (M.P.Irún et al., 2001). As shown in Figure 7Go (raw data in upper panel and apparent unfolded fraction after a three-state global analysis in lower panel), the fluorescence and near-UV CD curves display a temperature of mid-denaturation clearly lower than the absorbance and far-UV curves. This indicates that the two-state model does not apply to the thermal unfolding. We accordingly performed a global three-state analysis (see Methods), as described in Luo et al. (1995). In this analysis, the four curves of each protein variant are globally fitted to the equations of thermal unfolding, keeping {Delta}G, {Delta}Cp and Tm of the two equilibria (N/I and I/U) the same for the four curves. The results of such a fit for the wild-type protein are shown in Figure 7bGo and the calculated temperatures of mid-denaturation for the different protein variants are shown in Table VGo. The differences in Tm between the mutants with neutral hydrogen-bonding groups and the pseudo-wild-type protein are higher than those calculated from the simple two-state analysis of the fluorescence curves and are manifested as a stabilization of both the native state relative to the intermediate and of the intermediate relative to the unfolded state. Interestingly, the E61A and D126A control mutants display the same melting temperatures for the two equilibria as their corresponding reference proteins. Similarly, as already shown by the simple two-state fit, the D75A mutant is more stable than its reference. The results of the global three-state analysis thus reinforce the provisional conclusions derived from the simple two-state fits of the fluorescence thermal unfolding curves and agree well with the urea unfolding data.



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Fig. 7. Three-state global analysis of the thermal denaturation of wild-type apoflavodoxin followed by fluorescence, near-UV circular dichroism, far-UV circular dichroism and absorbance measurements. Fluorescence conditions: 2 µM apoflavodoxin in 50 mM MOPS, pH 7.0; ratio of emission at 320 and 360 nm. Near-UV absorbance and near-UV circular dichroism conditions (curves obtained simultaneously): 40 µM apoflavodoxin in 50 mM MOPS, pH 7.0; signal recorded at 291 nm. Far-UV circular dichroism conditions: 1 µM apoflavodoxin in 5 mM MOPS, pH 7.0 with 15 mM NaCl; ellipticity at 222 nm. (a) Raw data represented in arbitrary units for comparison; (b) global fit to a three-state model. Data are shown as apparent unfolded fractions. The solid lines are the best fit, with all thermodynamic parameters being the same for the four curves.

 

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Table V. Thermal denaturation of apoflavodoxin by fluorescence, near-UV CD, far-UV CD and absorbance: three-state global analysisa
 

    Discussion
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 Abstract
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 Materials and methods
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 Discussion
 References
 
Apoflavodoxin stabilization by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln

Inspired by the recent finding that neutral hydrogen bonds may be stronger than charged ones (Fernández-Recio et al., 1999Go), we wanted to test if proteins can be stabilized by neutralizing some of their charged hydrogen bonds. Although this could be studied in principle by measuring the stability of a given protein at different pH values, any change in pH will alter simultaneously many ionizing residues in almost every protein and interpretation of the results will be difficult. To avoid this problem, we devised a different strategy. Surface-exposed charged groups (aspartate and glutamate residues) that are hydrogen bonded to single partners in apoflavodoxin were mutated to their isosteric neutral amides (asparagine and glutamine), that retain hydrogen-bonding capability. The effect of the mutations on protein stability was then measured by urea and thermal denaturation. Three out of three engineered mutations consistently made apoflavodoxin more stable at both 25°C and higher temperatures, which is good evidence that the strategy is successful.

The stabilization is not due to removal of electrostatic repulsions and could be due to a higher strength of neutral hydrogen bonds relative to charged ones

When the stability of protein variants is compared, possible different secondary structure propensities of the residues involved should be considered. To avoid such complications, we selected mutations that are located either in loops (four cases) or in helices (two cases) where Asp and Asn, on the one hand, and Glu and Gln, on the other, have very similar propensities (see wwwbioq.unizar.es/helixsc.html).

Different explanations can be offered for the observed stabilization upon replacing charged hydrogen-bonded residues by their neutral amides, but the following two are the simplest. As mentioned above, there is recent evidence that neutral hydrogen bonds may sometimes be stronger that charged ones (Fernández-Recio et al., 1999Go). In this respect, mutations such as those we have introduced in apoflavodoxin are potentially stabilizing, provided that the hydrogen bond is retained. On the other hand, apoflavodoxin is a very acidic protein that at neutral pH bears a high excess negative charge. It is thus conceivable that lowering the negative excess charge by replacing aspartates/glutamates by their amides could have a general stabilizing effect that would explain the observed results. To investigate this second possibility, we studied the stability effect of three additional point mutations where aspartate or glutamate residues, solvent-exposed but not hydrogen-bonded, were replaced by alanine residues. Our data show that two of these three control mutants display essentially the same stability (both by urea and thermal denaturation) as wild-type. Based on this, a general stabilization of the apoflavodoxin structure by mutations involving non-hydrogen-bonded acidic groups can be ruled out. There is, however, one control mutant where a clear stabilizing effect is observed. Further analysis was performed to clarify if, in this mutant, the stabilization is indeed due to relief of electrostatic repulsions. If this were the case, the stabilizing effect would be significantly decreased at high ionic strength. We measured the stability of this mutant, relative to wild-type, in the same buffer but in the presence of 0.5 M choline chloride and the stabilization observed was very similar, which indicates that this mutation does not stabilize apoflavodoxin by removal of electrostatic repulsions but by some other, so far unknown, mechanism. In fact, 0.5 M choline chloride has an almost negligible effect on the stability of all the mutants analysed (relative to wild-type), including the three mutants involved in hydrogen bonding (Table IIIGo). The stabilizations we measure are therefore not due to removal of electrostatic repulsions. The simplest explanation that remains is that surface-exposed hydrogen bonds involving amides are stronger than those involving the corresponding carboxylic groups, in line with previous findings on a His/Tyr hydrogen bond (Fernández-Recio et al., 1999Go). This interpretation, however, relies on the assumption that the hydrogen bonds remain formed upon mutation, which is reasonable owing to the isosteric nature of the mutations, but confirmation will require further testing by X-ray analysis.

In practice, and whatever the cause, it seems that replacing hydrogen-bonded aspartate or glutamate residues at the surface of proteins by asparagine or glutamine, respectively, could be a simple approach towards increasing protein stability at 25°C and also towards increasing protein thermostability.


    Notes
 
1 To whom correspondence should be addressed. E-mail: jsancho{at}posta.unizar.es Back


    Acknowledgments
 
We acknowledge financial support from grants PB97-1027 (DGES, Spain) and P15/97 (CONSI+D, DGA, Spain). M.P. Irún was supported by an FPU fellowship (Spain).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received April 11, 2000; revised November 17, 2000; accepted December 20, 2000.