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
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Abstract |
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Keywords: 3D1D compatibility evaluation/human lysozyme/mutant stability/pseudo-energy potential/stability profile
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Introduction |
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To assure the physico-chemical meaning of the knowledge-based potentials, their applicability to protein stability analysis has been investigated (Miyazawa and Jernigan, 1994; Wang et al., 1996
; Gilis and Rooman, 1997
; Topham et al., 1997
). 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,
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,
GSPMP, have been weakly but significantly correlated with the experimentally determined differences in melting temperature between the mutant and wild-type proteins,
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 3D1D 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., 1992; Takano et al., 1995
, 1997a
, Takano et al., b
, 1998
, 1999
; Funahashi et al., 1996
; Yamagata et al., 1998
). 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., 1987; Ishikawa et al., 1993
; Akasako et al., 1995
; Mansfeld et al., 1997
). 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.
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Materials and methods |
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The changes in conformational stability due to single amino acid substitutions, GSPMP, of mutant human lysozymes were calculated by SPMP as described (Ota et al., 1995
). In SPMP, a pseudo-energy potential developed for evaluating structuresequence 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, 1993
; Ota et al., 1995
; Ota and Nishikawa, 1997
).
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The estimation of G for a replacement of residue X by residue Y is as follows.
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Mutant proteins
Mutagenesis, expression and purification of mutant human lysozymes were performed as described (Takano et al., 1995). 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., 1969
).
Differential scanning calorimetry (DSC)
Calorimetric measurements and data analyses were carried out as described (Takano et al., 1995). 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.
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X-Ray structural analysis
Mutant human lysozymes were crystallized as described (Takano et al., 1995; Yamagata et al., 1998
). 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, 1991). The data were processed with DENZO (Otwinowski, 1990
). 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, 1992) as described (Takano et al., 1995
; Yamagata et al., 1998
).
The coordinates of the mutant human lysozymes have been deposited in the Protein Data Bank, accession numbers 1b7l to 1b7s.
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Results |
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Human lysozyme consists of 130 amino acid residues. The stability changes upon mutation, G, of 130x19 mutant proteins were predicted by SPMP (Ota et al., 1995
) using the crystal structure of the wild-type protein (Takano et al., 1995
), as shown in Figure 1
. Figure 1
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 I
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 II
shows the mutant proteins chosen, the structural characteristics of the substituted residues in the wild-type structure, and the
GSPMP and
G values of four elements calculated from SPMP.
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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 2 shows typical excess heat capacity curves resulting from calorimetric recordings of the wild-type and mutant human lysozymes at pH 2.7. Table III
shows the denaturation temperature (Td), the calorimetric enthalpy (
Hcal), and the van't Hoff enthalpy (
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 IV
. 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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Data collection and refinement statistics for mutant human lysozymes, N27L, A32L, E35L, G37Q, Q58G, V74S, H78G and A96M, are summarized in Table V. The crystals of R50G and V100F were not obtained. The structures in the vicinity of the mutation sites are illustrated in Figure 3
.
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Discussion |
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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., 1992; Takano et al., 1997a
). 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., 1989; Eriksson et al., 1992
; Ishikawa et al., 1993
). However, unfavorable spatial contact and torsional strain cause the destabilization of a protein (Eriksson et al., 1993
). In the case of the mutant human lysozymes, A32L, A96M and V100F, the expected stabilization was not observed (Table IV
). 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 IV
). 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., 1987). 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 IV
). 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 c
). Hydrogen bonds between protein atoms and between protein and bound water molecules favorably contribute to protein stability (Myers and Pace, 1996
; Yamagata et al., 1998
; Takano et al., 1999
). 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, 1990). 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 IV
). 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 and
values (Table II
), known to be unfavorable conformations for amino acid residues, except in the case of glycine residues (Momany et al., 1975
). 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 IV
). 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
and
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., 1992); cavity-creating mutants, such as I23V and V93A (Takano et al., 1995
, 1997a
,Takano et al., b
); mutants related to side-chain hydrogen bonds, such as Y20F and S24A (Yamagata et al., 1998
; Takano et al., 1999
); 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
G values and the predicted values from SPMP is shown in Figure 4a
. The correlation coefficient is 0.48 (71 mutations), being slightly weaker than that of RNase H, 0.51 (96 mutations) (Ota et al., 1995
). 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 4b
. 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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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 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 4d
. It has been known that water molecules in the interior of a protein affect the stability favorably (Takano et al., 1997b
). 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, 1987). 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, 1997
; Samudrala and Moult, 1998
), 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., 1992
; Rice and Eisenberg, 1997
). 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, 1996
), 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 3D1D compatibility evaluation to create a table for the stability profile of mutant proteins, SPMP, showing changes in conformational stability, 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 3D1D compatibility evaluation.
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Acknowledgments |
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Notes |
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Received March 24, 1999; revised May 10, 1999; accepted May 11, 1999.