Experimental verification of the `stability profile of mutant protein' (SPMP) data using mutant human lysozymes

Kazufumi Takano, Motonori Ota1, Kyoko Ogasahara, Yuriko Yamagata2, Ken Nishikawa1 and Katsuhide Yutani3

Institute for Protein Research, Osaka University, Yamadaoka, Suita,Osaka 565-0871, 1 National Institute of Genetics, Yata, Mishima,Shizuoka 411-8540, 2 Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishikawa,K., 1995Go, J. Mol. Biol., 248, 733–738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudo-energy potential developed for evaluating structure–sequence compatibility in the structure prediction method, the 3D–1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability of SPMP. The thermodynamic parameters for denaturation and crystal structures of these mutant proteins were determined. One mutant protein was stabilized as expected, compared with the wild-type protein. However, the others were not stabilized even though the structural changes were subtle, indicating that SPMP overestimates the increase in stability or underestimates negative effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP. The correlation of the stability changes between the experiment and prediction depended on the types of substitution: there were some correlations for proline mutants and cavity-creating mutants, but no correlation for mutants related to side-chain hydrogen bonds. The present results may indicate some additional factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.

Keywords: 3D–1D compatibility evaluation/human lysozyme/mutant stability/pseudo-energy potential/stability profile


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Understanding the relationship between the amino acid sequence of a protein and its tertiary structure is one of the major goals of protein engineering (Gros and Tocchini-Valentin, 1994Go); however, this goal has not yet been achieved. One of the reasons is that a protein structure is prescribed not only by a part of the amino acid sequence but also by an extensive range of interactions (Minor and Kim, 1996). At present, a method of evaluating the compatibility of a sequence (1D) with a three-dimensional structure (3D) has attracted considerable attention as a prediction method for tertiary structures (Levitt, 1997Go; Marchler-Bauer et al., 1997Go). In this method, a sequence is mounted on a number of representative structures, each score of sequence–structure compatibility is estimated using empirically derived simple pseudo-energy potentials, and a best fit structure is suggested as a predicted structure.

To assure the physico-chemical meaning of the knowledge-based potentials, their applicability to protein stability analysis has been investigated (Miyazawa and Jernigan, 1994Go; Wang et al., 1996Go; Gilis and Rooman, 1997Go; Topham et al., 1997Go). Ota et al. (1995) have modified the pseudo-energy potential to create a table for the stability profile of mutant proteins, SPMP, showing changes in conformational stability, {Delta}{Delta}G, of possible point mutations not only at specific sites (e.g. buried/exposed sites) but also over the entire sequence by introducing the virtual denatured state. For ribonuclease H (RNase H), the stability changes in mutant proteins predicted, {Delta}{Delta}GSPMP, have been weakly but significantly correlated with the experimentally determined differences in melting temperature between the mutant and wild-type proteins, {Delta}Td, regardless of the site-specific features (e.g. buried/exposed sites). There is a question as to whether SPMP is applicable for proteins other than RNase H. To experimentally verify the applicability of SPMP to other proteins is also useful for the improvement in the quality of the pseudo-energy potential which is pivotal for the 3D–1D compatibility evaluation.

Human lysozyme has been used for the estimation of the effects of various factors, such as the hydrophobic effect and hydrogen bonds, on protein stability because it is possible to obtain thermodynamic parameters of the heat-denaturation process from differential scanning calorimetry (DSC) and obtain high resolution three-dimensional structures of the mutant proteins. More than 70 mutant proteins of human lysozyme have been investigated (Herning et al., 1992Go; Takano et al., 1995Go, 1997aGo, Takano et al., bGo, 1998Go, 1999Go; Funahashi et al., 1996Go; Yamagata et al., 1998Go). These data can be used to confirm the applicability of SPMP. Unfortunately, most of the mutant proteins reported are destabilized compared with the wild-type protein.

Protein stabilization is one of the main goals of protein engineering. Although numerous studies have been reported for mutant proteins, there are only a few cases that have stabilized proteins as expected (Matthews et al., 1987Go; Ishikawa et al., 1993Go; Akasako et al., 1995Go; Mansfeld et al., 1997Go). SPMP produces a table predicting changes in stability due to point mutations, and shows the mutations most likely to increase stability. SPMP will prove useful if it can accurately predict the stabilized mutant proteins.

In this paper, the mutant human lysozymes, the stabilization of which was predicted by SPMP, were constructed and measurements of thermal stability and X-ray structural analysis were carried out. The result showed that only one mutant protein was stabilized as expected. The reason why SPMP reached estimations different from the experimental results was examined, and the improvement of SPMP was discussed, using the results of structural analysis of the mutant proteins and the data from previous studies of other mutant human lysozymes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Stability profile of mutant proteins (SPMP)

The changes in conformational stability due to single amino acid substitutions, {Delta}{Delta}GSPMP, of mutant human lysozymes were calculated by SPMP as described (Ota et al., 1995Go). In SPMP, a pseudo-energy potential developed for evaluating structure–sequence compatibility in the structure prediction method was employed, consisting of four elements: side-chain packing, hydration, local conformation and hydrogen-bonding efficiency of the backbone (Nishikawa and Matsuo, 1993Go; Ota et al., 1995Go; Ota and Nishikawa, 1997Go).

where {Delta}{Delta}GSP, {Delta}{Delta}GHyd, {Delta}{Delta}GLC and {Delta}{Delta}GHB represent the changes in Gibbs energy due to side-chain packing, hydration, local conformation and hydrogen-bonding efficiency of the backbone, respectively. The side-chain packing function is a Sippl-type pairwise function, considering the distance between the side-chains and the interacting directions (Sippl, 1990Go), but not considering in detail the conformation of the side-chains. The hydration function is based on the partitioning of an amino acid residue type into the surface or interior of a globular protein (Nishikawa and Ooi, 1980Go; Rose and Roy, 1980Go): in the interior of a protein, a hydrophobic amino acid is preferred rather than a hydrophilic one, but on the surface of a protein, the reverse is true. The local conformation function is a potential estimated from the frequencies of an amino acid residue observed in a conformational state. The hydrogen-bonding efficiency of the backbone function is given to the pair of proton donors (oxygen atoms) and acceptors (nitrogen atoms) in the backbone atoms, depending on the preference for hydrogen bond formation between two amino acid residue types. The pseudo-energy potential provides a fitness score for each residue type of a site in a native structure.

The estimation of {Delta}{Delta}G for a replacement of residue X by residue Y is as follows.


where G(X) and <G(X)> represent the fitness score for residue X of a site in a native structure calculated by the pseudo-energy potential and the average G(X) of all sites in the considered protein, respectively, corresponding with the Gibbs energy in the native and denatured states for residues X, GN(X) and GD(X), respectively.

Mutant proteins

Mutagenesis, expression and purification of mutant human lysozymes were performed as described (Takano et al., 1995Go). The mutant proteins of human lysozyme, of which stabilization was expected by SPMP, N27L, A32L, E35L, R50G, Q58G, V74S, H78G, A96M and V100F, and of which destabilization was expected, G37Q, were constructed. The concentration of the mutant proteins was spectrophotometrically determined using E1%(1 cm) = 25.65 at 280 nm (Parry et al., 1969Go).

Differential scanning calorimetry (DSC)

Calorimetric measurements and data analyses were carried out as described (Takano et al., 1995Go). For measurement, the DASM4 adiabatic microcalorimeter equipped with an NEC personal computer was used. Data analysis was done using Origin software (MicroCal, Inc., Northampton, MA).

The thermodynamic parameters for denaturation as a function of temperature were calculated using the following equations.



where {Delta}Cp values are assumed to be independent of temperature (Privalov and Khechinashvili, 1974Go).

X-Ray structural analysis

Mutant human lysozymes were crystallized as described (Takano et al., 1995Go; Yamagata et al., 1998Go). All crystals belong to space group P212121 with a crystal form identical to that of the wild-type and of most mutant proteins.

For A96M, the data set was collected at 100 K using synchrotron radiation at the Photon Factory (Tsukuba) on beam lines 6A with a Weissenberg camera (Sakabe, 1991Go). The data were processed with DENZO (Otwinowski, 1990Go). For V74S, the data set was collected at 100 K using synchrotron radiation at the SPring-8 (Harima) on beam line 41XU (Proposal No. 1997B0159-NL-np). For N27L, A32L, E35L, G37Q and H78G, the data set was collected at 100 K by the oscillation method on the Rigaku R-AXIS IV imaging plate mounted on the Rigaku RU300 rotating anode X-ray generator. For Q58G, the data set was collected at 288 K by the oscillation method on the Rigaku R-AXIS IIc imaging plate mounted on the Rigaku RU300 rotating anode X-ray generator. The data were processed with software provided by Rigaku.

The structures of mutant proteins were solved by the isomorphous method. The structures were refined with the program X-PLOR (Brunger, 1992Go) as described (Takano et al., 1995Go; Yamagata et al., 1998Go).

The coordinates of the mutant human lysozymes have been deposited in the Protein Data Bank, accession numbers 1b7l to 1b7s.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prediction of stability change of mutant human lysozymes by SPMP and selection of mutant proteins examined

Human lysozyme consists of 130 amino acid residues. The stability changes upon mutation, {Delta}{Delta}G, of 130x19 mutant proteins were predicted by SPMP (Ota et al., 1995Go) using the crystal structure of the wild-type protein (Takano et al., 1995Go), as shown in Figure 1Go. Figure 1Go indicates that there are more substitutions expected to destabilize (on the left side of the Figure) than those expected to stabilize (on the right side of the Figure). Table IGo lists the best 50 substitutions expected to be more stable than wild-type. In this study, nine mutant proteins expected to increase in stability were selected. The nine substitutions were at different positions and were predicted to stabilize for different reasons: for A96M, A32L and V100F, the main contribution to the stability was side-chain packing energy; for E35L, N27L and V74S, it was hydration and side-chain packing energies; for H78G, Q58G and R50G, it was local conformation energy. Furthermore, one mutant protein, G37Q, expected to decrease in stability by local conformation energy was also examined as a control. Table IIGo shows the mutant proteins chosen, the structural characteristics of the substituted residues in the wild-type structure, and the {Delta}{Delta}GSPMP and {Delta}{Delta}G values of four elements calculated from SPMP.



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Fig. 1. `Stability profile of mutant protein' of human lysozyme. This diagram indicates how each of the 20 amino acid residues prefers a given site environment in comparison with the preference of the wild-type protein. White circles and plus symbols denote the hydrophobic (Ile, Leu, Phe, Val, Trp, Met, Cys, Ala and Tyr) and the other residues, respectively. The substitutions examined in this study are represented by solid circles and labeled.

 

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Table I. The best 50 substitutions of human lysozyme expected to increase stability relative to the wild-type protein calculated from SPMP
 

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Table II. The mutant human lysozymes chosen in this study, the structural characteristics of the substituted residues in the wild-type protein, and the {Delta}{Delta}G values (kJ/mol) calculated from SPMP
 
Differential scanning calorimetry (DSC) of mutant human lysozymes

In order to determine the thermodynamic parameters of denaturation of mutant human lysozymes, DSC measurements were done at acidic pHs between 2.4 and 3.3 where the denaturation of the proteins is completely reversible. Figure 2Go shows typical excess heat capacity curves resulting from calorimetric recordings of the wild-type and mutant human lysozymes at pH 2.7. Table IIIGo shows the denaturation temperature (Td), the calorimetric enthalpy ({Delta}Hcal), and the van't Hoff enthalpy ({Delta}HvH) of each measurement for the mutant proteins. Because the secretion in yeast of N27L was lower than that of the wild-type protein and the protein concentration of N27L at the measurement was low, only the peak temperature of denaturation was determined. The thermodynamic parameters of denaturation at a constant temperature, 64.9°C, and pH 2.7 were calculated using these data as shown in Table IVGo. The stability of the mutant proteins, except for Q58G and V100F, was comparable with that of the wild-type protein. Q58G was stabilized as expected by SPMP, but V100F was largely destabilized compared with the wild-type protein.





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Fig. 2. Typical excess heat capacity curves of the wild-type, V100F and Q58G human lysozymes. The increments of excess heat capacity are 10 kJ/mol K.

 

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Table III. Thermodynamic parameters for denaturation of mutant human lysozymes at different pH values
 

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Table IV. Thermodynamic parameters for denaturation of mutant human lysozymes at the denaturation temperature (64.9°C) of the wild-type protein at pH 2.7
 
X-Ray structural analysis of mutant human lysozymes

Data collection and refinement statistics for mutant human lysozymes, N27L, A32L, E35L, G37Q, Q58G, V74S, H78G and A96M, are summarized in Table VGo. The crystals of R50G and V100F were not obtained. The structures in the vicinity of the mutation sites are illustrated in Figure 3Go.


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Table V. X-Ray data collection and refinement statistics of mutant human lysozymes
 


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Fig. 3. ORTEP (Johnson, 1976) views showing the structure in the vicinity of the mutation sites. Panels (a) to (h) represent N27L, A32L, E35L, G37Q, Q58G, V74S, H78G and A96M, respectively. The wild-type (open bonds) and mutant (filled bonds) structures are superimposed. Solvent water molecules are drawn as open circles (wild-type) and crossed circles (mutant). The broken lines indicate hydrogen bonds.

 
The structural changes due to substitution were not large for all the mutant proteins examined. The side chains of N27 and E35 in the wild-type structure form hydrogen bonds with S24 and a water molecule, respectively. In the mutant structures of N27L and E35L, each hydrogen bond disappeared on substitution (Figure 3a and cGo). The substitution of small to large side-chains in the interior of the proteins A32L and A96M caused small structural changes in the side-chains surrounding the mutation sites (Figure 3b and hGo). For the mutant proteins, G37Q, Q58G and H78G, changes in the backbone conformation were not observed (Figure 3d, e and gGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutant human lysozymes expected to increase stability

Nine mutant human lysozymes expected to increase stability according to the calculation of SPMP were examined in this study. One was stabilized as expected compared with the wild-type protein, and it is the most stable one among the mutant proteins investigated so far (Herning et al., 1992Go; Takano et al., 1997aGo). However, the other eight mutants showed similar stabilities to the wild-type protein or were destabilized in spite of small structural changes due to substitution. The results suggest that SPMP overestimates the increase in stability or underestimates negative effects due to substitution.

Side-chain packing: A32L, A96M and V100F It has been known that the improvement of side-chain packing, such as cavity-filling substitution in the interior of a protein, causes an increase in conformational stability (Karpusas et al., 1989Go; Eriksson et al., 1992Go; Ishikawa et al., 1993Go). However, unfavorable spatial contact and torsional strain cause the destabilization of a protein (Eriksson et al., 1993Go). In the case of the mutant human lysozymes, A32L, A96M and V100F, the expected stabilization was not observed (Table IVGo). The stabilization by the increase in side chain size must be compensated by unfavorable effects, because SPMP does not consider in detail the conformation of the side chains. The stability of A32L and A96M was comparable with that of the wild-type protein, but V100F was largely destabilized (Table IVGo). One of the reasons might be the differences in volume and shape of the cavity in each mutation site. The results suggest that compensation for unfavorable packing effects in the case of small to large mutations should be included in the calculation of SPMP.

Hydration: N27L, E35L and V74S In the interior of a protein, the conformational stability generally correlates with hydrophobicity of the amino acid residues without structural hindrance (Yutani et al., 1987Go). N27 and E35 in human lysozyme are buried polar residues. The substitution of these residues by non-polar ones are expected to increase stability. However, the stability of the N27L and E35L mutant human lysozymes was comparable with that of wild-type (Table IVGo). This must be caused by the fact that the substitutions of N27 and E35 removed their side-chain hydrogen bonds, which are not included in the calculation of SPMP. N27 and E35 in the wild-type structure form hydrogen bonds with S24 and a water molecule in the interior of the protein, respectively (Figure 3a and cGo). Hydrogen bonds between protein atoms and between protein and bound water molecules favorably contribute to protein stability (Myers and Pace, 1996Go; Yamagata et al., 1998Go; Takano et al., 1999Go). Yamagata et al. (1998) have estimated the contribution of an intramolecular hydrogen bond to protein stability to be about 8 kJ/mol. In the case of N27L and E35L, the stabilization by the improvement in hydrophobicity compensated for the destabilization due to removal of the hydrogen bonds. Therefore, it is necessary for SPMP to include the effect due to changes in side-chain hydrogen bonds.

On the surface of a protein, polar residues are more ubiquitous than non-polar ones. In the hydration function of SPMP, polar residues are preferred rather than non-polar ones on the protein surface. A substitution on the protein surface theoretically has no effect on the stability, because the site is exposed to solvent in both the native and denatured states, although reverse hydrophobic effects are possible (Pakula and Sauer, 1990Go). V74 in human lysozyme is a non-polar residue exposed to the solvent. The substitution of V74 by polar residues was expected to increase the stability according to SPMP, but there was actually little effect on stability (Table IVGo). Thus, the hydration function on the surface of a protein in SPMP needs to be improved.

Local conformation: R50G, Q58G, H78G and G37Q R50, Q58, H78 and G37 in the human lysozyme structure have positive {Phi} and {Psi} values (Table IIGo), known to be unfavorable conformations for amino acid residues, except in the case of glycine residues (Momany et al., 1975Go). The prediction due to SPMP shows that the mutant proteins of R50G, Q58G and H78G are stabilized and that of G37Q is destabilized compared with the wild-type protein. However, the substitutions of R50, H78 and G37 had little effect on stability, although Q58G was stabilized as expected (Table IVGo). The difference in the results of these mutant proteins corresponded with that of the accessibility of the substituted residues: Q58 is buried but the others are exposed to the solvent. These experimental results can be explained by the following: (i) flexibility, (ii) reverse hydrophobic effects or (iii) conformational entropy. (i) The positive angles of {Phi} and {Psi} in a flexible region might not be unfavorable for structural stability, because the backbone in the interior of a protein is rigid, but that on the surface is flexible. (ii) Pakula and Sauer (1990) have reported that proteins can be stabilized by reverse hydrophobic effects when mutations are introduced on the surface of proteins. In the case of R50G and H78G, the reverse hydrophobic effects might compensate for the increase in the stability due to mutation, resulting in little change in the stability. (iii) Mutation of a large side chain to a small one reduces conformational entropy in a denatured state, resulting in stabilization of the protein. However, such a mutation on the protein surface also might reduce entropy in the native state, resulting in no effect on the stability. On the other hand, the increase in the stability of Q58G, 7.8 kJ/mol, is comparable with the stabilization due to the entropic effect of the replacement of Q by G, 8.1 kJ/mol, estimated by Pickett and Sternberg (1993). These results suggest that the local conformation function is applicable to the mutation in the interior of a protein.

Analysis of other reported mutants of human lysozymes

The stability and structures of more than 70 mutant human lysozymes have already been reported: proline mutants, such as P71G and A47P (Herning et al., 1992Go); cavity-creating mutants, such as I23V and V93A (Takano et al., 1995Go, 1997aGo,Takano et al., bGo); mutants related to side-chain hydrogen bonds, such as Y20F and S24A (Yamagata et al., 1998Go; Takano et al., 1999Go); and a series of mutants at the same position, such as I56M, I56F and I56T (J.Funahashi, K.Takano, Y.Yamagata and K.Yutani, unpublished results). These data can be used for confirming the reliability of SPMP. The correlation for all mutant human lysozymes between the experimental {Delta}{Delta}G values and the predicted values from SPMP is shown in Figure 4aGo. The correlation coefficient is 0.48 (71 mutations), being slightly weaker than that of RNase H, 0.51 (96 mutations) (Ota et al., 1995Go). The difference is due to the difference in type of substitution used between human lysozyme and RNase H. In the case of mutant human lysozymes, there are many mutants related to side-chain hydrogen bonds which are not included in the calculation of SPMP. The correlation coefficient for the hydrogen-bond mutants was –0.04 (22 mutations), as shown in Figure 4bGo. These results suggest that hydrogen bonds contribute to the stability of a protein, a factor that should be included in the calculation of SPMP.



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Fig. 4. Correlations between {Delta}{Delta}GSPMP and {Delta}{Delta}GExperiment for mutant human lysozymes, (a) all mutant proteins, (b) mutant proteins related to side-chain hydrogen bonds, (c) proline mutant proteins, and (d) cavity-creating mutant proteins except for those introducing water molecules in the cavities created by substitutions. In panel (c), filled and open squares represent the mutant proteins substituted in the interior of the protein and on the surface of the protein, respectively.

 
For proline mutants, the values in SPMP mainly come from the effect of local conformation. There is a good correlation of {Delta}{Delta}G between experimental and predicted values for the proline mutants as shown in Figure 4cGo, suggesting the accuracy of the local conformation function. However, although the calculations of the proline mutations in the interior of a protein were coincident with the experiments (filled squares in Figure 4cGo), the mutations on the protein surface had little effect on stability (open squares in Figure 4cGo), similar to the cases of the glycine mutants, R50G, Q58G, H78G and G37Q, as described above. These results indicate that the local conformation function is useful for the mutations in the interior of a protein, but not for those on the surface of a protein, because of the effects of flexibility or side-chain entropy as discussed above.

The side-chain packing and hydration functions in SPMP are the main contributors to the stability for the cavity-creating mutations such as Ile to Val. There was a clear correlation (correlation coefficient of 0.59 for 31 points) of {Delta}{Delta}G between experimental and predicted values for these mutants, except for those containing water molecules introduced in the cavities created by the substitutions, as shown in Figure 4dGo. It has been known that water molecules in the interior of a protein affect the stability favorably (Takano et al., 1997bGo). Furthermore, in the case of small to large substitutions in the interior of the protein (cavity-filling mutants) such as A96M, SPMP does not compensate for the unfavorable packing effects, as described above. Therefore, it seems that the side-chain packing and hydration functions in SPMP are useful for the mutations in the interior of a protein, which are not characterized by unfavorable spatial contacts and torsional strain, and structural changes such as the introduction of water molecules.

Improvement of SPMP

The structure stability analysis by SPMP should be improved. First, the introduction of a more detailed treatment for side-chains, such as a rotamer library (Ponder and Richards, 1987Go). In this study, it was revealed that a simple representation of the side chain was inadequate, especially to estimate the cavity-filling mutants or the side-chain hydrogen-bonding mutants. If side chains with higher resolutions are employed (Melo and Feytmans, 1997Go; Samudrala and Moult, 1998Go), illegal collisions between side chains could be detected and the cavity-filling mutants would be more precisely evaluated. The formation of hydrogen bonds between side chains could be also considered. Secondly, hydration and local-conformation functions may be joined (Bowie et al., 1992Go; Rice and Eisenberg, 1997Go). If the two functions are treated more clearly, the illegal dihedral angle of a given amino acid on the exposed site could be allowed. These modifications assessing their applicability should be also tried. Furthermore, the estimation of reverse hydrophobicity appears to be one of the most serious problems in the methods using the knowledge-based potential, because the apparent interactions are intrinsically counted in the energy function transformed from the known structures; exposed hydrophobic residues are penalized by the knowledge-based potential because they are rarely observed (Thomas and Dill, 1996Go), even if it is not disadvantageous for them in view of the free energy of denaturation. This should be addressed in the future.

Conclusions

Ota et al. (1995) have modified the pseudo-energy potential used in the 3D–1D compatibility evaluation to create a table for the stability profile of mutant proteins, SPMP, showing changes in conformational stability, {Delta}{Delta}G, of possible point mutations. In this study, the verification of SPMP was experimentally tested by applying it to mutant human lysozymes. From the analysis, it was shown that the factors not minutely considered in SPMP lowered its efficiency. If the improvements in SPMP are made, SPMP might prove useful for protein engineering. Moreover, the improvements will also permit progress with the 3D–1D compatibility evaluation.


    Acknowledgments
 
We thank Takeda Chemical Ind., Ltd, for providing the plasmid pGEL125. This work was supported in part by a Grant-in-Aid for special project research from the Ministry of Education, Science and Culture of Japan (K.Y. and Y.Y.), by Fellowships from the Japan Society for the Promotion of Science for Young Scientists (K.T.), by the Japan Space Utilization Promotion Center (K.Y.), and by the Sakabe project of TARA, University of Tsukuba (Y.Y.).


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Akasako,A., Haruki,M., Oobatake,M. and Kanaya,S. (1995) Biochemistry, 34, 8115–8122.[ISI][Medline]

Bowie,J.U., Luthy,R. and Eisenberg,D. (1992) Science, 253, 164–170.[ISI]

Brunger,A.T. (1992) X-PLOR Manual, Ver. 3.1. Yale University, New Haven, CT.

Eriksson,A., Baase,W., Zhang,X.-J., Heinz,D.W., Blaber,M., Baldwin,E.P. and Matthews,B.W. (1992) Science, 255, 178–183.[ISI][Medline]

Eriksson,A., Baase,W. and Matthews,B.W. (1993) J. Mol. Biol., 229, 747–769.[ISI][Medline]

Funahashi,J., Takano,K., Ogasahara,K., Yamagata,Y. and Yutani,K. (1996) J. Biochem., 120, 1216–1223.[Abstract]

Gilis,D. and Rooman,M. (1997) J. Mol. Biol., 272, 276–290.[ISI][Medline]

Gros,F. and Tocchini-Valentin,G.G. (1994) Nature, 369, 11–12.[ISI][Medline]

Herning,T., Yutani,K., Inaka,K., Kuroki,R., Matsushima,M. and Kikuchi,M. (1992) Biochemistry, 31, 7077–7085.[ISI][Medline]

Ishikawa,K., Nakamura,H., Morikawa,K. and Kanaya,S. (1993) Biochemistry, 32, 6171–6178.[ISI][Medline]

Johnson,C.K. (1976) ORTEPII, Oak Ridge National Laboratory, TN.

Karpusas,M., Baase,W.A., Matsumura,M. and Matthews,B.W. (1989) Proc. Natl Acad. Sci. USA, 86, 8237–8241.[Abstract]

Levitt,M. (1997) Proteins, suppl., 1, 92–104.

Mansfeld,J., Vriend,G., Dijkstra,B.W., Veltman,O.R., Burg,B.V., Venema,G., Ulbrich-Hofmann,R. and Eijsink,V.G.H. (1997) J. Biol. Chem., 272, 11152–11156.[Abstract/Free Full Text]

Marchler-Bauer,A., Levitt,M. and Bryant,S.H. (1997) Proteins, suppl., 1, 83–91.

Matthews,B.W., Nicholson,H. and Becktel,W.J. (1987) Proc. Natl Acad. Sci. USA, 84, 6663–6667.[Abstract]

Melo,F. and Feytmans,E. (1997) J. Mol. Biol., 267, 207–222.[ISI][Medline]

Minor,D.L.,Jr. and Kim,P.S. (1996) Nature, 380, 730–734.[ISI][Medline]

Miyazawa,S. and Jernigan,R.L. (1994) Protein Engng, 7, 1209–1220.[Abstract]

Momany,F.A., McGuire,R.F., Burgess,A.W. and Scheraga,H.A. (1975) J. Phys. Chem., 79, 2361–2381.[ISI]

Myers,J.K. and Pace,C.N. (1996) Biophys. J., 71, 2033–2039.[Abstract]

Nishikawa,K. and Matsuo,Y. (1993) Protein Engng, 6, 811–820.[Abstract]

Nishikawa,K. and Ooi,T. (1980) Int. J. Peptide Protein Res., 16, 19–32.[ISI][Medline]

Ota,M., Kanaya,S. and Nishikawa,K. (1995) J. Mol. Biol., 248, 733–738.[ISI][Medline]

Ota,M. and Nishikawa,K. (1997) Protein Engng, 10, 339–351.[Abstract]

Otwinowski,Z. (1990) DENZO Data Processing Package. Yale University, New Haven, CT.

Pakula,A.A. and Sauer,R.T. (1990) Nature, 344, 363–364.[ISI][Medline]

Parry,R.M., Chandan,R.C. and Shahani,K.M. (1969) Arch. Biochem. Biophys., 130, 59–65.[ISI][Medline]

Pickett,S.D. and Sternberg,M.J. (1993) J. Mol. Biol., 231, 825–839.[ISI][Medline]

Ponder,J.W. and Richards,F.M. (1987) J. Mol. Biol., 193, 775–791.[ISI][Medline]

Privalov,P.L. and Khechinashvili,N.N. (1974) J. Mol. Biol., 86, 665–684.[ISI][Medline]

Rice,D.W. and Eisenberg,D. (1997) J. Mol. Biol., 267, 1026–1038.[ISI][Medline]

Rose,G.D. and Roy,R. (1980) Proc. Natl Acad. Sci. USA, 77, 4643–4647.[Abstract]

Sakabe,N. (1991) Nucl. Instr. Methods Phys. Res., A303, 448–463.[ISI]

Samudrala,R. and Moult,J. (1998) J. Mol. Biol., 275, 895–916.[ISI][Medline]

Sippl,M.J. (1990) J. Mol. Biol., 213, 859–883.[ISI][Medline]

Takano,K., Ogasahara,K., Kaneda,H., Yamagata,Y., Fujii,S., Kanaya,E., Kikuchi,M., Oobatake,M. and Yutani,K. (1995) J. Mol. Biol., 254, 62–76.[ISI][Medline]

Takano,K. Yamagata,Y., Fujii,S. and Yutani,K. (1997a) Biochemistry, 36, 688–698.[ISI][Medline]

Takano,K., Funahashi,J., Yamagata,Y., Fujii,S. and Yutani,K. (1997b) J. Mol. Biol., 274, 132–142.[ISI][Medline]

Takano,K., Yamagata,Y. and Yutani,K. (1998) J. Mol. Biol., 280, 749–761.[ISI][Medline]

Takano,K., Yamagata,Y., Kubota,M., Funahashi,J., Fujii,S. and Yutani,K. (1999) Biochemistry, 38, 6623–6629.[ISI][Medline]

Thomas,P.D. and Dill,K.A. (1996) J. Mol. Biol., 257, 457–469.[ISI][Medline]

Topham,C.M., Srinivasan,N. and Blundell,T.L. (1997) Protein Engng, 10, 7–21.[Abstract]

Wang,Y., Lal,L., Li,S., Han,Y. and Tang,Y. (1996) Protein Engng, 9, 479–484.[Abstract]

Yamagata,Y., Kubota,M., Sumikawa,Y., Funahashi,J., Takano,K., Fujii,S. and Yutani,K. (1998) Biochemistry, 37, 9355–9362.[ISI][Medline]

Yutani,K., Ogasahara,K., Tsujita,T. and Sugino,Y. (1987) Proc. Natl Acad. Sci. USA, 84, 4441–4444.[Abstract]

Received March 24, 1999; revised May 10, 1999; accepted May 11, 1999.