Protein Engineering, Vol. 14, No. 2, 127-134,
February 2001
© 2001 Oxford University Press
Are the parameters of various stabilization factors estimated from mutant human lysozymes compatible with other proteins?
1 Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
The various factors which contribute to protein stability have been extensively examined using mutant proteins, but the same kinds of substitutions have given different results depending on the substitution sites. Recently, the contributions of some stabilization factors have been quantitatively derived as parameters by a unique equation, considering the conformational changes due to the mutations using mutant human lysozymes [Funahashi et al. (1999) Protein Eng. 12, 841–850]. To evaluate these parameters estimated from the mutant human lysozymes, stability–structure datasets for the mutant T4 lysozymes were selected. The stabilities for the mutant T4 lysozymes could be roughly estimated using these parameters. Notable differences between the estimated and experimental stabilities were caused by the uncertainty in part of the structures due to some Arg and Lys residues fluctuating on the surface of the T4 lysozyme. Excluding these atoms from the estimation gave a good correlation between the estimated and experimental stabilities. These results suggest that the parameters of the various stabilization factors derived from the mutant human lysozymes are compatible with the mutant T4 lysozymes, although they should be improved with respect to some points using more information.
Keywords: estimation of protein stability/lysozyme/protein stability/protein stabilization factors/protein structure
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
The stability of proteins is balanced by the combination of the stabilization and destabilization factors, such as the hydrophobic effect, hydrogen bonding and entropic effects (Pace et al., 1996
). The magnitude of each factor is not very small, but their combination results in only a 40 kJ/mol conformational stability of the proteins (Privalov and Gill, 1988; Dill, 1990). To understand the mechanism of protein stability, many factors that contribute to it have been studied using mutant proteins in which substitutions would affect each stabilizing factor. However, the same kinds of substitutions have given different results. For instance, studies of hydrophobic mutant proteins (e.g. Yutani et al., 1977, 1987; Kellis et al., 1988, 1989; Matsumura et al., 1988; Shortle et al., 1990; Eriksson et al., 1992; Jackson et al., 1993; Takano et al., 1995, 1997a, Takano et al., b, 1998) have shown that the hydrophobic residues in a protein contribute to the stability but the contribution differs depending on the differences in the environments surrounding the substitution residues and structural changes due to the mutations. Studies of mutant proteins in which a hydrogen-bonded residue is substituted with one incapable of hydrogen bonding have also shown that the loss in stability per deleted hydrogen bond varies depending on the location (e.g. Green et al., 1992; Serrano et al., 1992; Blaber et al., 1993a,b; Byrne et al., 1995; Hammen et al., 1995; Matthews, 1995; Yamagata et al., 1998; Takano et al., 1999a,b). Thus, it is difficult to estimate independently the contribution of each stabilization factor to protein stability. Moreover, a substitution may affect not only the mutation site but also other parts of the protein far from the site, although the structural changes are not large (Takano et al., 1997a).
It has been proposed that the stability of each mutant human lysozyme can be represented by a unique equation, considering the structural changes due to mutations (Funahashi et al., 1999). In the study, by a least-squares fit of the experimental Gibbs energy changes upon denaturation (
Gexp) of 36 mutant human lysozymes to the equation, the contribution of each stabilization factor, such as the hydrophobic effect, hydrogen bonding and water molecule introduced in the interior of a protein, to the stability has been estimated. Moreover, the contribution of the hydrogen bonds to the protein stability has been divided into three terms depending on the type of its partner using the mutant human lysozymes by a similar process (Takano et al., 1999a, b). The results of these reports suggest the possibility that the contribution of each stabilization factor to protein stability can be independently and quantitatively estimated using this process. The question is whether the contribution of various stabilization factors to the stability calculated from the mutant human lysozymes is compatible with other proteins.
The T4 lysozyme (164 residues) is a good candidate for the confirmation because its mutants have been extensively constructed and their stabilities and structures have been examined (Matthews, 1995). In the present study, using 54 mutant human lysozymes and 56 mutant T4 lysozymes, the contribution of several stabilization factors to protein stability was analyzed in relation to the changes in the stability and the structure due to the substitutions and the results obtained from both the human and T4 lysozymes will be comparatively discussed. Here, the hydrophobic effect, conformational entropy of a side chain of a substituted residue, hydrogen bond, water molecules introduced in the interior of a protein due to substitution, propensities of the 
-helix and ß-sheet of substituted residue and cavity volume were considered as the stabilization factors.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Mutant human lysozymes
All mutant human lysozymes used in this study have been previously examined by us (Yutani et al., 1999). The stability was measured by differential scanning calorimetry (DASM4) in the acidic region (pH 2.5–3.3) where the heat denaturation of the human lysozyme is completely reversible. The changes in the denaturation Gibbs energy (G) between the wild-type and the mutant proteins were compared at the denaturation temperature of the wild-type protein at pH 2.7 (64.9°C). All of the crystal structures of the mutant human lysozymes were studied by X-ray crystallography as described and deposited in the Protein Data Bank. The crystals of most mutants belong to the same crystal form as the wild-type protein (Takano et al., 1995, 1997a, Takano et al., b, 1998, 1999a, Takano et al., b; Funahashi et al., 1996, 1999; Yamagata et al., 1998). However, the crystal forms of some mutants differ from that of the wild-type protein (Osserman et al., 1969; Funahashi et al., 1999; Takano et al., 1999a). There are two molecules in the asymmetric unit and the values of the total molecular accessible surface area (ASA) for these two molecules differ from each other. Because the ASA value is important for our analysis as described below, the mutant proteins whose crystal forms differ from that of the wild-type protein were not selected for the analysis. In addition, the hydrophobic effect, the conformational entropy of the side chain of the substituted residue, the hydrogen bond, the water molecules introduced in the interior of the protein due to substitution, the propensities of the -helix and ß-sheet of the substituted residue and the cavity volume were considered as the stabilization factors in this study. Therefore, the mutant proteins, which would be affected by the other stabilization factors, such as the steric hindrance and the salt bridge, were also not included in these analyses.
The 54 selected mutant human lysozymes were I23V, I56V, I59V, I89V, I106V (Takano et al., 1995); I56T (Funahashi et al., 1996); I23A, I56A, I59A, I89A, I106A, I59G (Takano et al., 1997a); V2A, V74A, V93A, V99A, V100A, V110A, V121A, V125A (Takano et al., 1997b); I23V-3ss, I56V-3ss, I89V-3ss, I106V-3ss, V2A-3ss, V74A-3ss, V110A-3ss, V121A-3ss, V125A-3ss (Takano et al., 1998); Y20F, Y38F, Y45F, Y54F, Y63F, Y124F (Yamagata et al., 1998); S24A, S36A, S51A, S61A, S80A (Takano et al., 1999a); T11A, T11V, T40A, T43A, T43V, T52A, T70V (Takano et al., 1999b); I56L, I56M, I59L, I59M, I59S and I59T (Fuanahashi et al., 1999).
Mutant T4 lysozymes
The mutant T4 lysozymes were selected from the studies of Matthews and co-workers using the same method as for the mutant human lysozymes described above. In their studies, the mutant T4 lysozymes were constructed using the gene for the wild-type T4 lysozyme or for a pseudo-wild-type T4 lysozyme, Cys54Thr/Cys97Ala, as a template (Matsumura and Matthews, 1989; Eriksson et al., 1992). Therefore, each mutant T4 lysozyme was compared with the corresponding wild-type protein. The stabilities of the mutant T4 lysozymes were measured from the CD data at 223 nm taken as a function of temperature. The thermodynamic parameters were compared at 53.5 or 51.8°C, which are the denaturation temperature of the wild-type or pseudo-wild-type T4 lysozyme, respectively, and at pH 3.0, where the heat denaturation of the T4 lysozyme is reversible (Matsumura and Matthews, 1989). We used the crystal structures of the mutant proteins from the Protein Data Bank.
The 56 selected mutant T4 lysozymes were T157A, T157V, T157G, T157S (Alber et al., 1987); I3V (Matsumura et al., 1988); N55G (Nicholson et al., 1989); T152S (Dao-pin et al., 1991); V131A/N132A (Zhang et al., 1991); L46A, L99A, L118A, L121A, L133A, F153A, L99A/F153A (Eriksson et al., 1992); S44A (Heinz et al., 1992); T59A, T59G, T59V, T59S (Bell et al., 1992); F153L, F153M (Eriksson et al., 1993); T26A, T151S (Pjura and Matthews, 1993); V75T, V87T, V149T (Blaber et al., 1993a); S44G, V131A, V131G, V131S (Blaber et al., 1993b); M120A, N116A, T115A, S117A, T115A/S117A (Blaber et al., 1995); I78A, L91A, F104M, M106A (Gassner et al., 1996); I17A, I27A, I29A, I50A, I58A, I100A, V87A, V103A, V111A, V149A, F67A, L84A, M6A (Xu et al, 1998); M102A, F104A and L133G (Baldwin et al., 1998).
Calculation of ASA values and cavity volume
The accessible surface area (ASA) values and cavity volume of the proteins were calculated by the previously described procedure of Connolly (1993) with probes of 1.4 and 1.2 Å, respectively (Takano et al., 1997b, 1998; Funahashi et al., 1999). The ASA values of the denatured state were calculated using an actual polypeptide of three residues including the mutation site in the middle (Oobatake and Ooi, 1993; Takano et al., 1997a). The water molecule located in the interior of a protein was not removed from the structure during the calculation of the cavity volume.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
The contribution of various stabilization factors to the stabilities of the mutant human lysozymes
The stabilization factors were proposed and examined based on mutagenesis studies (Eriksson et al., 1992; Pace, 1992; Serrano et al., 1992; Shirley et al., 1992; Buckle et al., 1993; Byrne et al., 1995; Yu et al., 1995; Myers and Pace, 1996; Pace et al., 1996; Xu et al., 1998). Recently, it has been proposed that the changes in stability of each mutant human lysozyme are represented by a unique equation, considering the conformational changes due to the mutations (Funahashi et al., 1999). Changes in the hydrophobic effect, conformational entropy, hydrogen bonding and water molecule introduced in the interior of a protein have been considered as the stabilization factors. The difference in the Gibbs energy changes upon denaturation between the wild-type and mutant proteins (G) is expressed by the following equation (Funahashi et al., 1999):
 | (1) |
where GHP, Gconf, GHB and GH2O represent the differences in G between the wild-type and each mutant protein due to the hydrophobic effect, conformational entropy of the substituted residue, forming or removing hydrogen bonding and introducing water molecules, respectively. Each G can be expressed by each parameter in terms of the conformational change:
 | (2) |
 | (3) |
 | (4) |
 | (5) |
where ASANP and ASAP represent the differences in the ASA of the non-polar (C and S) and polar atoms (O and N) of all residues in a protein upon denaturation, respectively; ASA means the difference in ASA between the wild-type and each mutant protein; Sconf is the conformational entropy defined by Doig and Sternberg (1995); rHB is the length of the hydrogen bonds; and NH2O is the changes in the number of water molecules introduced by the substitutions. The parameters of , ß, 
and 
have been estimated to be 0.178 kJ/mol.Å2, –0.013 kJ/mol.Å2, 15.53 kJ Å/mol and –7.79 kJ/mol, respectively, by a least-squares fit of each G (Equation 1
) to the experimental G values using the stability–structure database of the mutant human lysozymes (Funahashi et al., 1999).
Coefficients of ASA ( and ß) in Equation 2 are equivalent to the atomic solvation parameter (ASP) (Eisenberg and McLachlan, 1986). The ASP value represents the contribution of each protein atom to the solvation Gibbs energy related to the accessibility of the atom to the solvent. The value of = 15.53 in Equation 4 suggests that if a hydrogen bond with a length of 3.0 Å is removed by substitution, the mutant protein should be destabilized by 5.1 kJ/mol. This value is comparable to the values estimated by other investigators. Myers et al. (1997), for example, concluded that hydrogen bonds stabilize proteins and the average net stabilization is 4.2–8.4 kJ/mol. The value means that when one water molecule is newly introduced into the interior of a mutant protein, the protein is destabilized by 7.8 kJ/mol due to an entropic effect. Dunitz (1994) estimated that the decrease in entropy caused by the transfer of a water molecule from the solvent to the interior of a protein corresponds to a maximum Gibbs energy cost of 10 kJ/mol at 65°C.
The hydrogen bonding parameter has been revised by Takano et al. (1999b), who classified the hydrogen bond terms (Equation 4) into the following three classes:
 | (6) |
where [pp], [pw] and [ww] mean the intramolecular, protein–water and water–water hydrogen bonds, respectively. The values of [pp], [pw] and [ww] derived from the mutant human lysozymes are 25.63, 15.60 and 14.91 kJ Å/mol, respectively. These values show that the contributions of the 3 Å intra-, intermolecular and water–water hydrogen bonds to protein stability are 8.5, 5.2 and 4.9 kJ/mol, respectively, indicating the different contribution of each hydrogen bond.
In addition to these stabilization factors, the propensity term of the secondary structure (Gpro) has been added to Equation 1 (Funahashi et al., 2000):
 | (7) |
where pro[] and pro[ß] are the -helix and the ß-sheet propensities, respectively, of the residue defined by Chou and Fasman (1978) [revised by Koehl and Levitt (1999)]. The values of 
[] and [ß] derived from the mutant human lysozymes are 5.07 and 2.31 kJ/mol, respectively. This assumes that the substitution with the residues observed more frequently on the secondary structure contributes more strongly to the protein stability (Takano et al., 1997a). These [] and [ß] values represent that, for example, the Val to Ala substitution in the -helix stabilizes the mutant proteins by 2.9 kJ/mol and the same substitution in the ß-sheet destabilizes it by 1.7 kJ/mol.
In the present study, one more stabilization factor was newly added to Equation 1. The contribution of the changes in the cavity volume to the protein stability has been reported (Eriksson et al., 1992; Matthews, 1996; Xu et al., 1998). This term can be represented as follows:
 | (8) |
where Vcav represents the changes in the cavity volume due to the substitution. This coefficient has been estimated to be –0.100 kJ/mol.Å3 using some mutant T4 lysozymes (Eriksson et al., 1992).
Each parameter could be determined in several steps depending on the mutant nature, as reported previously (Funahashi et al., 1999; Takano et al., 1999b). To estimate the unified contribution of each stabilization factor to the stability of the human lysozyme, furthermore, the parameters , ß, [pp], [pw], [ww], , [], [ß] and 
were determined at the same time, using the equation
 | (9) |
 |
 |
 |
 |
A least-squares fit of the experimental G values (Gexp) to Equation 9 using the stability–structure database of 54 mutant human lysozymes examined in our previous studies (Takano et al., 1995, 1997a, Takano et al., b, 1998, 1999a, Takano et al., b; Funahashi et al., 1996, 1999; Yamagata et al., 1998) gave = 0.154, ß = –0.026, [pp] = 25.70, [pw] = 14.13, [ww] = 17.13, = –8.45, [] = 5.09, [ß] = 2.05 and = –0.052. Figure 1 shows the correlation between Gexp and the estimated G values (Gest) using these parameters. There was a good correlation between them (SD = 2.2 kJ/mol). Table I lists the contribution of each factor to the 54 mutant stabilities. The changes in stability were mainly attributed to GHP and GHB. The entropic penalty of introducing water molecule(s) in the interior of the protein (GH2O) was large, but it was compensated by GHB because the introduced water molecule forms hydrogen bond(s). The contribution of the other stabilization factors was somewhat small but never negligible.
|
|
Table II
summarizes the parameters of each stabilization factor derived from our previous studies and those from the present study using the mutant human lysozymes. They were slightly different from each other because the types of stabilization factors considered and the determination procedures (step by step and at the same time) were also different from each other. Fortunately, however, the contribution of each stabilization factor derived from the present study to the stability of the mutant human lysozymes coincided with our previous studies, within experimental error. This indicates that the Gibbs energy components are additive and their parameters are reliable.
|
Estimation of stabilities for mutant T4 lysozymes using the parameters derived from mutant human lysozymes
It was examined whether the parameters of the various stabilization factors estimated from the mutant human lysozymes are compatible with the mutant T4 lysozymes. Using the parameters of the mutant human lysozymes, changes in the stability due to mutations for the 56 mutant T4 lysozymes were estimated. Figure 2 shows the correlation between Gexp and Gest. The error for the estimated values was large (SD = 10.0 kJ/mol). To understand the origin of this large deviation, the structural changes of six mutant T4 lysozymes, I27A, I58A, N116A, V131A/N132A, V149A and V149T, whose differences between Gexp and Gest were large (Figure 2), were re-examined. As a result, the values of ASAP from I27A, I58A, N116A, V131A/N132A, V149A and V149T were –93.4, –123,5, –261.5, 88.8, –106.3 and –143.1 Å2, respectively. The ASAP values versus the residue number for the six mutant proteins are shown in Figure 3, where the residues with large values are marked. Almost all of them were Lys and Arg and largely exposed. The B-factor for the polar atoms of the side chain in those residues was extremely high, nearly 100 Å2, in the wild-type structure. Because thermal vibrations are large on the surface of a protein, the B-factor of these residues would be reported to be large, suggesting an uncertainty in their coordinates in the crystal structure. The number of Arg and Lys is 15 and 14, respectively, in the wild-type T4 lysozyme. Most of them are protruding from the surface of the protein and have a large B-factor. On the other hand, the B-factor for all atoms in the structure of the wild-type human lysozyme is less than 60 Å2. To minimize the effect due to the thermal vibrations for the mutant T4 lysozymes on the estimation, the ASA values were recalculated by excluding the atoms whose B-factor were large in the wild-type structure. Figure 4 shows the correlation between Gexp and Gest estimated using the recalculated ASA values. In these cases, the atoms whose B-factor were greater than 90 Å2 (Figure 4a) and 70 Å2 (Figure 4b) were excluded from the recalculation. The number of atoms whose B-factors were greater than 70 Å2 was 54 out of 1292 and B-factors for 39 of them were greater than 90 Å2. The errors between Gexp and Gest became small [(a) SD = 8.1; (b) 7.6 kJ/mol] when the atoms whose coordinates were uncertain were excluded. Since Gest was sensitive to ASA, it is necessary to use the structural data carefully. The errors between Gexp and Gest are still large, but minimization of such thermal vibrations gave a better correlation between Gexp and Gest. This result suggests that the notable differences between Gexp and Gest were caused by the uncertainty in the structures due to some atoms fluctuating on the surface of the T4 lysozyme.
|
|
|
The points at issue and future prospects of this analysis
The parameters derived from mutant human lysozymes were roughly compatible with the mutant T4 lysozymes. Here, the parameter of each stabilization factor was derived from only the dataset of the mutant T4 lysozymes. A least-squares fit of Gexp to Equation 9 for 48 mutant T4 lysozymes gave = 0.122, ß = 0.049, [pp] = 16.44, [pw] = 16.36, [] = 0.79 and = –0.088. In this case, because the dataset contains a limited number of mutant proteins substituted at the ß-sheet or with water(s) introduced in the interior of the protein, Gpro[ß], GH2O and GHB[ww] in Equation 9 were not considered. To minimize the uncertainty in part of the structure, the recalculated ASA values were used as mentioned above. Figure 5 shows the correlation between Gexp and Gest estimated using the dataset of T4 lysozyme. The correlation between them (SD = 3.9 kJ/mol) was better than that when Gest was estimated using the parameters from the human lysozyme. When a total of 110 mutant proteins of both lysozymes (54 mutant human lysozymes and 56 mutant T4 lysozymes) were used, the parameters were = 0.146, ß = 0.021, [pp] = 22.08, [pw] = 9.13, [ww] = 7.70, = –4.51, [] = 3.33, [ß] = 0.11 and = –0.073 (Figure 6; SD = 4.3 kJ/mol). Table III summarizes the parameters of each stabilization factor derived from human, T4 and both lysozymes. The parameters were essentially similar between human and T4 lysozymes. However, the magnitude of the parameters for human and T4 lysozymes was somewhat different from each other and the differences between Gexp and Gest were still large, which are the points at issue which should be improved.
|
|
|
In this study, we assumed extended structures to be that in the denatured state. The ASA values in the denatured state, however, might be differently affected, depending on the local sites. In fact, it has been reported that, in some cases, it is better for the estimation of the mutant protein stability to assume native-like structures to be that in the denatured state (Sugita et al., 1998
). On the other hand, the frequency of the amino acids observed in the -helix is different depending on the location of the mutation site in the -helix (Richardson and Richardson, 1988). Therefore, the parameters of [] should be divided into several terms, such as into the N-terminal, center or C-terminal parts of the -helix in a great store of dataset. Furthermore, there were important stabilization factors which were not considered in the present analysis, such as changes in the steric hindrance and in the electrostatic interaction. For example, the differences between Gexp and Gest of the I56F, I59F and I59Y human lysozymes were 8–18 kJ/mol, although those of the other mutant human lysozymes were maximized at 4.2 kJ/mol (Funahashi et al., 1999). Because positions 56 and 59 are completely buried in the interior of the human lysozyme, these differences seem to be mainly caused by the occurrence of steric hindrance. It is important that possible structural specificities (including denatured state) are classified in detail and assigned to each parameter. The differences in the parameters between human and T4 lysozymes should decrease if appropriate parameters are sufficiently supplemented. Conclusions
The present results suggest that the analysis proposed in this study is appropriate for a basic understanding of protein stability. However, it is essential for further analysis to introduce many more parameters, if necessary, and to accumulate much more stability–structure datasets for the mutant proteins. In this case, structural data should be of high quality because the parameters are sensitive to the conformational changes such as ASA. Improving the analysis at these points leads to the parameters of the stabilization factors that can precisely estimate changes in protein stability from its structural change. These parameters also provide a guide for enhancing the protein stability due to amino acid substitutions, which is one of the objectives of protein engineering.
|
Notes |
|---|
2 To whom correspondence should be addressed. E-mail: yutani{at}protein.osaka-u.ac.jp
|
Acknowledgments |
|---|
This work was supported in part by Fellowships from the Japan Society for the Promotion of Science for Young Scientists (J.F. and K.T.) and by a grant-in-aid for Scientific Research on Priority Areas (c) `Genome Information Science' from the Ministry of Education, Science and Culture of Japan (K.Y.).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Alber,T., Dao-pin,S., Wilson,K., Wozniak,J.A., Cook,S.P. and Matthews,B.W. (1987) Nature, 330, 41–46.[Medline]
Baldwin,E., Baase,W.A., Zhang,X.-J., Feher,V. and Matthews,B.W. (1998) J. Mol. Biol., 277, 467–485.[Web of Science][Medline]
Bell,J.A., Becktel,W.J., Sauer,W., Baase,W.A. and Matthews,B.W. (1992) Biochemistry, 31, 3590–3596.[Medline]
Blaber,M., Lindstrom,J.D., Gassner,N., Xu,J., Heinz,D.W. and Matthews,B.W. (1993a) Biochemistry, 32, 11363–11373.[Medline]
Blaber,M., Zhang,X.-J. and Matthews.B.W. (1993b) Science, 260, 1637–1640.
Blaber,M., Baase,W.A., Gassner,N. and Matthews,B.W. (1995) J. Mol. Biol., 246, 317–330.[Web of Science][Medline]
Buckle,A.M., Henrick,K. and Fersht,A.R. (1993) J. Mol. Biol., 234, 847–860.[Web of Science][Medline]
Byrne,M.P., Manuel,R.L., Lowe,L.G. and Stites,W.E. (1995) Biochemistry, 34, 13949–13960.[Medline]
Chou,P.Y. and Fasman,G.D. (1978) Adv. Enzymol., 47, 45–148.
Connolly,M.L. (1993) J. Mol. Graphics, 11, 139–141.[Web of Science][Medline]
Dao-pin,S., Alber,T., Basse,W.A., Wozniak,J.A. and Matthews,B.W. (1991) J. Mol. Biol., 221, 647–667.[Web of Science][Medline]
Dill,K.A. (1990) Biochemistry, 29, 7133–7155.[Medline]
Doig,A.J. and Sternberg,M.J.E. (1995) Protein Sci., 4, 2247–2251.[Web of Science][Medline]
Dunitz,J.D. (1994) Science, 264, 670.
Eisenberg,D. and McLachlan,A.D. (1986) Nature, 319, 199–203.[Medline]
Eriksson,A.E., Baase,W.A., Zhang,X.-J., Heinz,D.W., Blaber,M., Baldwin,E.P. and Matthews,B.W. (1992) Science, 225, 178–183.
Eriksson,A.E., Baase,W.A. and Matthews,B.W. (1993) J. Mol. Biol., 229, 747–769.[Web of Science][Medline]
Funahashi,J., Takano,K., Ogasahara,K., Yamagata,Y. and Yutani,K. (1996) J. Biochem., 120, 1216–1223.
Funahashi,J., Takano,K., Yamagata,Y. and Yutani,K. (1999) Protein Eng., 12, 841–850.
Funahashi,J., Takano,K., Yamagata,Y. and Yutani,K. (2000) Biochemistry, 39, 14448–14456.[Medline]
Gassner,N.C., Baase,W.A. and Matthews,B.W. (1996) Proc. Natl Acad. Sci. USA, 93, 12155–12158.
Green, S.M., Meeker,A.K. and Shortle,D. (1992) Biochemistry, 31, 5717–5728.[Medline]
Hammen,P.K., Scholtz,J.M., Anderson,J.W., Waygood,E.B. and Klevit,R.E. (1995) Protein Sci., 4, 936–944.[Web of Science][Medline]
Heinz,D.W., Basse,W.A. and Matthews,B.W. (1992) Proc. Natl Acad. Sci. USA, 89, 3751–3755.
Jackson,S.E., Moracci,M., elMasry,N., Johnson,C.M. and Fersht,A.R. (1993) Biochemistry, 32, 11259–11269.[Medline]
Kellis,J.T.,Jr., Nyberg,K., S
ali,D. and Fersht,A.R. (1988) Nature, 333, 784–786.[Medline]
Kellis,J.T.,Jr, Nyberg,K. and Fersht,A.R. (1989) Biochemistry, 28, 4914–4922.[Medline]
Koehl,P. and Levitt,M. (1999) Proc. Natl Acad. Sci. USA, 96, 12524–12529.
Matsumura,M. and Matthews,B.W. (1989) Science, 243, 792–794.
Matsumura,M., Becktel,W.J. and Matthews,B.W. (1988) Nature, 334, 406–410.[Medline]
Matthews,B.W. (1995) Adv. Protein Chem., 46, 249–278.[Web of Science][Medline]
Matthews,B.W. (1996) FASEB J., 10, 35–41.[Abstract]
Myers,J.K. and Pace,C.N. (1996) Biophys. J., 71, 2033–2039.[Web of Science][Medline]
Myers,J.K., Pace,C.N. and Scholtz,J.M. (1997) Proc. Natl Acad. Sci. USA, 94, 2833–2837.
Nicholson,H., Soderlind,E., Tronrud,D.E. and Matthews,B.W. (1989) J. Mol. Biol., 221, 647–667.
Oobatake,M. and Ooi,T. (1993) Prog. Biophys. Mol. Biol., 59, 237–284.[Web of Science][Medline]
Osserman,E.F., Cole,S.J., Swan,I.D.A. and Blake,C.C.F. (1969) J. Mol. Biol., 46, 211–212.[Web of Science][Medline]
Pace,C.N. (1992) J. Mol. Biol., 226, 29–35.[Web of Science][Medline]
Pace,C.N., Shirley,B.A., McNutt,M. and Gajiwala,K. (1996) FASEB J., 10, 75–83.[Abstract]
Pjura, P. and Matthews,B.W. (1993) Protein Sci., 2, 2226–2232.[Web of Science][Medline]
Privalov,P.L. and Gill,S.J. (1988) Adv. Protein Chem., 39, 191–234.[Web of Science][Medline]
Richardson,J.S. and Richardson,D.C. (1988) Science, 240, 1648–1652.
Serrano,L., Kellis,J.T., Cann,P., Matouschek,A. and Fersht,A.R. (1992) J. Mol. Biol., 224, 783–804.[Web of Science][Medline]
Shirley,B.A., Stanssens,R., Hahn,U. and Pace,C.N. (1992) Biochemistry, 31, 725–732.[Medline]
Shortle,D., Stites,W.E. and Meeker,A.K. (1990) Biochemistry, 29, 8033–8041.[Medline]
Sugita,Y., Kitao,A. and Go,N. (1998) Folding Des., 3, 173–181.[Web of Science][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.[Web of Science][Medline]
Takano,K., Funahashi,J., Yamagata,Y., Fujii,S. and Yutani,K. (1997a) J. Mol. Biol., 274, 132–142.[Web of Science][Medline]
Takano,K., Yamagata,Y., Fujii,S. and Yutani,K. (1997b) Biochemistry, 36, 688–698.[Medline]
Takano,K., Yamagata,Y. and Yutani,K. (1998) J. Mol. Biol., 280, 749–761.[Web of Science][Medline]
Takano,K., Yamagata,Y., Kubota,M., Funahashi,J., Fujii,S. and Yutani,K. (1999a) Biochemistry, 38, 6623–6629.[Medline]
Takano,K., Yamagata,Y., Funahashi,J., Hioki,Y., Kuramitsu,S. and Yutani,K. (1999b) Biochemistry, 38, 12698–12708.[Medline]
Xu,J., Baase,W.A., Baldwin,E. and Matthews,B.W. (1998) Protein Sci., 7, 158–177.[Web of Science][Medline]
Yamagata,Y., Kubota,M., Sumikawa,Y., Funahashi,J., Takano,K., Fujii,S. and Yutani,K. (1998) Biochemistry, 37, 9355–9362.[Medline]
Yu,M.-H., Weissman,J.S. and Kim.P.S. (1995) J. Mol. Biol., 249, 388–397.[Web of Science][Medline]
Yutani, K., Ogasahara, K., Sugino, Y. and Matsushiro,A. (1977) Nature, 267, 274–275.[Medline]
Yutani,K., Ogasahara,K., Tsujita,T. and Sugino,Y. (1987) Proc. Natl Acad. Sci. USA, 84, 4441–4444.
Yutani,K., Takano,K. and Funahashi,J. (1999) In Kuwajima,K. and Arai,M. (eds), Old and New Views of Protein Folding. Elsevier, Tokyo, pp. 175–184.
Zhang,X.-J., Basse,W.A. and Matthews,B.W. (1991) Biochemistry, 30, 2012–2017.[Medline]
Received November 7, 2000; revised November 28, 2000; accepted November 30, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Kang, G. Chen, and G. Xiao Robust prediction of mutation-induced protein stability change by property encoding of amino acids Protein Eng. Des. Sel., February 1, 2009; 22(2): 75 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Asada, M. Sawano, K. Ogasahara, J. Nakamura, M. Ota, C. Kuroishi, M. Sugahara, K. Yutani, and N. Kunishima Stabilization Mechanism of the Tryptophan Synthase {alpha}-Subunit from Thermus thermophilus HB8: X-Ray Crystallographic Analysis and Calorimetry J. Biochem., October 1, 2005; 138(4): 343 - 353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Taka, K. Ogasahara, J. Jeyakanthan, N. Kunishima, C. Kuroishi, M. Sugahara, S. Yokoyama, and K. Yutani Stabilization Due to Dimer Formation of Phosphoribosyl Anthranilate Isomerase from Thermus thermophilus HB8: X-Ray Analysis and DSC Experiments J. Biochem., May 1, 2005; 137(5): 569 - 578. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Funahashi, Y. Sugita, A. Kitao, and K. Yutani How can free energy component analysis explain the difference in protein stability caused by amino acid substitutions? Effect of three hydrophobic mutations at the 56th residue on the stability of human lysozyme Protein Eng. Des. Sel., September 1, 2003; 16(9): 665 - 671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Kaushik and R. Bhat Why Is Trehalose an Exceptional Protein Stabilizer?: AN ANALYSIS OF THE THERMAL STABILITY OF PROTEINS IN THE PRESENCE OF THE COMPATIBLE OSMOLYTE TREHALOSE J. Biol. Chem., July 11, 2003; 278(29): 26458 - 26465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Takano, Y. Yamagata, and K. Yutani Buried water molecules contribute to the conformational stability of a protein Protein Eng. Des. Sel., January 1, 2003; 16(1): 5 - 9. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Takano and K. Yutani A new scale for side-chain contribution to protein stability based on the empirical stability analysis of mutant proteins Protein Eng. Des. Sel., August 1, 2001; 14(8): 525 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ota, Y. Isogai, and K. Nishikawa Knowledge-based potential defined for a rotamer library to design protein sequences Protein Eng. Des. Sel., August 1, 2001; 14(8): 557 - 564. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||