Structural and Thermodynamic Responses of Mutations at a Ca2+ Binding Site Engineered into Human Lysozyme

Structural determinants of Ca2+binding sites within proteins typically comprise several acidic residues in appropriate juxtaposition. Three residues (Ala-83, Gln-86, and Ala-92) in human lysozyme are characteristically mutated to Lys, Asp, and Asp, respectively, in natural Ca2+ binding lysozymes and α-lactalbumins. The effects of these mutations on the stability and Ca2+ binding properties of human lysozyme were investigated using calorimetry and were interpreted with crystal structures. The double mutant, in which Glu-86 and Ala-92 were replaced with Asp, clearly showed Ca2+ binding affinity, whereas neither point mutant showed Ca2+ affinity, indicating that both residues are essential. The further mutation of Ala-83 → Lys did not affect the Ca2+ binding of the double mutant. The point mutations Ala-83 → Lys and Glu-86 → Asp did not affect the stability, whereas the mutation Ala-92 → Asp was about 1.3 kcal/mol less stable. Structural analyses showed that both Asp-86 and Lys-83 were exposed to solvent. Side chains of Asp-86 and Asp-91 were rotated in opposite directions about χ1 angle, as if to reduce the electrostatic repulsion. The charged amino acids at the Ca2+ binding site did not significantly affect stability of the protein, possibly because of the local conformational change of the side chains. Structural determinants of Ca2+binding sites within proteins typically comprise several acidic residues in appropriate juxtaposition. Three residues (Ala-83, Gln-86, and Ala-92) in human lysozyme are characteristically mutated to Lys, Asp, and Asp, respectively, in natural Ca2+ binding lysozymes and α-lactalbumins. The effects of these mutations on the stability and Ca2+ binding properties of human lysozyme were investigated using calorimetry and were interpreted with crystal structures. The double mutant, in which Glu-86 and Ala-92 were replaced with Asp, clearly showed Ca2+ binding affinity, whereas neither point mutant showed Ca2+ affinity, indicating that both residues are essential. The further mutation of Ala-83 → Lys did not affect the Ca2+ binding of the double mutant. The point mutations Ala-83 → Lys and Glu-86 → Asp did not affect the stability, whereas the mutation Ala-92 → Asp was about 1.3 kcal/mol less stable. Structural analyses showed that both Asp-86 and Lys-83 were exposed to solvent. Side chains of Asp-86 and Asp-91 were rotated in opposite directions about χ1 angle, as if to reduce the electrostatic repulsion. The charged amino acids at the Ca2+ binding site did not significantly affect stability of the protein, possibly because of the local conformational change of the side chains. Calcium binding proteins are known to take part in several important functions in biological systems (1Levine B.A. Williams R.J.P. Cheung W.-Y. Calcium and Cell Function. II. Academic Press, New York1982: 1-38Google Scholar). Ca2+ binding sometimes accompanies conformational changes, which are considered to be responsible for biological functions such as signal transduction and the formation of macromolecular complexes. Ca2+ binding sites usually consist of several acidic residues chelating to bound Ca2+ in a pentagonal bipyramidal manner (2Strynadka N.C.J. James M.N.G. Curr. Opin. Struct. Biol. 1991; 1: 905-914Crossref Scopus (50) Google Scholar). The close locations of acidic residues in a Ca2+ binding site should negatively affect protein stability due to charge repulsion between the acidic residues. How does the introduction of charged residues within a calcium binding site affect the stability and Ca2+ binding behavior? There are some reports describing the effect of mutations within Ca2+ binding sites on the stability of the protein (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar, 4Haezebrouck P. De Baetselier A. Joniau M. Van Deal H. Rosenberg S. Hanssens I. Protein Eng. 1993; 6: 643-649Crossref PubMed Scopus (20) Google Scholar). Our approach in this paper is to introduce the minimum perturbation by amino acid replacement and determine the effect of these mutations on the stability and the Ca2+ binding function using calorimetry. Furthermore, these effects are interpreted in terms of the structural information from x-ray crystallography. For this purpose, we chose human lysozyme because lysozymes are known to be a suitable model for studying protein function and stability (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar, 5Kuroki R. Taniyama Y. Seko C. Nakamura H. Kikuchi M. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6903-6907Crossref PubMed Scopus (104) Google Scholar, 6Kuroki R. Inaka K. Taniyama Y. Kidokoro S. Matsushima M. Kikuchi M. Yutani K. Biochemistry. 1992; 31: 8323-8328Crossref PubMed Scopus (72) Google Scholar, 7Kuroki R. Nitta K. Yutani K. J. Biol. Chem. 1992; 267: 24297-24301Abstract Full Text PDF PubMed Google Scholar). More than 90 sequences of chicken type lysozymes and α-lactalbumins are in the sequence data base. Among these lysozymes, several have been found to have Ca2+ binding ability (8Nitta K. Tsuge H. Sugai S. Shimazaki K. FEBS Lett. 1987; 223: 405-408Crossref PubMed Scopus (66) Google Scholar, 9Nitta K. Tsuge H. Shimazaki K. Sugai S. Biol. Chem. Hoppe-Seyler. 1988; 369: 671-675Crossref PubMed Scopus (51) Google Scholar, 10Godovac-Zimmerman J. Conti A. Napolitano L. Biol. Chem. Hoppe-Seyler. 1988; 369: 1109-1115Crossref PubMed Scopus (37) Google Scholar, 11Jollès P. Lysozymes: Model Enzymes in Biochemistry and Biology. Birkhäuser Verlag, Basel1996Crossref Google Scholar). Three residues (Ala-83, Gln-86, and Ala-92) in human lysozyme are usually mutated to Lys, Asp, and Asp, respectively, in natural Ca2+ binding lysozymes and α-lactalbumins as shown in Fig.1. We have already found that only the double mutation (Gln-86 → Asp and Ala-92 → Asp) in human lysozyme resulted in the formation of a Ca2+ binding site (5Kuroki R. Taniyama Y. Seko C. Nakamura H. Kikuchi M. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6903-6907Crossref PubMed Scopus (104) Google Scholar). The precise analyses of the stability (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar, 5Kuroki R. Taniyama Y. Seko C. Nakamura H. Kikuchi M. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6903-6907Crossref PubMed Scopus (104) Google Scholar, 6Kuroki R. Inaka K. Taniyama Y. Kidokoro S. Matsushima M. Kikuchi M. Yutani K. Biochemistry. 1992; 31: 8323-8328Crossref PubMed Scopus (72) Google Scholar) and the Ca2+affinity (7Kuroki R. Nitta K. Yutani K. J. Biol. Chem. 1992; 267: 24297-24301Abstract Full Text PDF PubMed Google Scholar) using calorimetry have also been performed, and the high resolution structural data of the wild type (12Artymiuk P.J. Blake C.C.F. J. Mol. Biol. 1981; 152: 737-762Crossref PubMed Scopus (263) Google Scholar) and Ca2+binding mutant (13Inaka K. Kuroki R. Kikuchi M. Matsushima M. J. Biol. Chem. 1991; 266: 20666-20671Abstract Full Text PDF PubMed Google Scholar) lysozymes from x-ray crystallography are available. Here we show the effect of the subsequent mutations (Gln-86 → Asp, Ala-92 → Asp, and Ala-83 → Lys) on the Ca2+ binding properties, conformational stabilities, and tertiary structures of these mutants. It was found that both aspartic acids (Asp-86 and Asp-86) are essential for Ca2+ binding. The introduction of an aspartic acid at the Ala-92 position resulted in destabilization of lysozyme, whereas the introduction of an aspartic acid at the Gln-86 position or the introduction of positive charge the Ala-83 position did not affect the stability. The observed instability with the Ala-92 → Asp mutation is proposed to result from the difference in the hydration effect.DISCUSSIONSeveral acidic residues accompanying some basic residues are characteristically associated to make a Ca2+ binding site in Ca2+ binding proteins. Acidic residues are found to directly chelate to Ca2+ in a pentagonal bipyramidal manner (2Strynadka N.C.J. James M.N.G. Curr. Opin. Struct. Biol. 1991; 1: 905-914Crossref Scopus (50) Google Scholar, 13Inaka K. Kuroki R. Kikuchi M. Matsushima M. J. Biol. Chem. 1991; 266: 20666-20671Abstract Full Text PDF PubMed Google Scholar), and some basic residues located in the vicinity of Ca2+ binding sites appear to provide a counter ion charge to the chelating residues. Three chelating aspartic acids, Asp-86, Asp-91, and Asp-92 and the positively charged Lys-83 are conserved in Ca2+ binding lysozymes and α-lactalbumins. Because Asp-91 already exists in wild type human lysozyme (Fig. 1), the effect of the three mutations, Gln-86 → Asp, Ala-92 → Asp, and Ala-83 → Lys on the stability and affinity of Ca2+ was investigated using calorimetry. In Table V, the effect of the mutations on the stability is summarized, in which ΔΔG at 80 °C was calculated according to Becktel and Shellman (27Becktel W.J. Schellman J.A. Biopolymers. 1987; 26: 1859-1877Crossref PubMed Scopus (940) Google Scholar) by assuming that ΔS and ΔC p values of Ca2+ bound and unbound mutants are the same as those of the holo-Q86D/A92D and the wild type lysozymes, respectively, as reported previously (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar). In the presence of 10 mm Ca2+, the mutant A83K/Q86D/A92D showed about 3.6 kcal/mol stabilization, which is similar to that of Q86D/A92D lysozyme. Other mutants, and lysozymes, did not show in the presence of to Schellman J.A. Biopolymers. Scopus Google Scholar, J.A. Biopolymers. Scopus Google Scholar), the of binding such as a Ca2+ binding should the stability of the protein in the presence of the of 10 which is about in of the protein should result in of the In both and mutant lysozymes, was observed in the presence of Ca2+ the mutants Q86D/A92D and that both the and mutants not have Ca2+ affinity, which is with the of the binding as in Table also that both acidic residues Asp-86 and Asp-92 are essential for Ca2+ binding in the mutant human lysozyme. In the of Ca2+, we the effect of the mutation on the stability of lysozyme. The stability of lysozyme was the same as that of the wild type lysozyme, indicating affect of the mutation from Gln-86 to The lysozyme was about 1.3 kcal/mol less than the wild which is similar to the and lysozymes, indicating that the mutation Ala-83 to Lys did not affect the stability the mutation Ala-92 to Asp resulted in 1.3 kcal/mol for the of the mutant human lysozymes and values were to 80 The ΔS and ΔC p for Ca2+ bound mutants in 10 mm were to be the same as those of the and the ΔS and ΔC p for Ca2+ unbound mutants and were to be the same as those of the wild type lysozyme reported reported reported (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google ΔΔG values were to 80 The ΔS and ΔC p for Ca2+ bound mutants in 10 mm were to be the same as those of the (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar), and the ΔS and ΔC p for Ca2+ unbound mutants and were to be the same as those of the wild type lysozyme (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar). in a the stability in terms of the structural high resolution structural data were from x-ray crystallography. these aspartic side chains are located within to chelate Ca2+ and lysozymes in and the introduction of aspartic acid at position resulted in a of the side the of the site as if to reduce the charge repulsion to Asp-91 lysozyme in The charged of Asp-86, and Lys-83 are located and 10 from Asp-91 in the wild type lysozyme, the mutations Gln-86 → Asp or Ala-83 → Lys did not affect the stability. It that a charged a Ca2+ binding site does not the protein stability. a of the side be to reduce the repulsion between the because of the It been reported that an ion located on the of a protein does not affect the stability of the protein U. H. Biochemistry. 1991; PubMed Scopus Google Scholar). the a destabilization was observed for the mutation Ala-92 → for this destabilization is the difference in hydration between the and Asp side chains The tertiary of the mutant that the side of Asp-92 is and the side was similar to those of the holo-Q86D/A92D lysozyme to and M. J. Biochemistry. 1989; Scopus Google Scholar, M. Mol. Biol. 1993; PubMed Scopus Google Scholar), the difference in hydration between and is calculated to be about kcal/mol at 80 Because Asp-92 in the mutant and Ala-92 in the wild type structures are with the of these amino acids calculated to be less than the difference in hydration is considered to be the part of the instability kcal/mol in Table observed in the mutants The than effect on stability be by the binding of ion to Asp-92 in the as in the crystal structures of the and lysozyme. Calcium binding proteins are known to take part in several important functions in biological systems (1Levine B.A. Williams R.J.P. Cheung W.-Y. Calcium and Cell Function. II. Academic Press, New York1982: 1-38Google Scholar). Ca2+ binding sometimes accompanies conformational changes, which are considered to be responsible for biological functions such as signal transduction and the formation of macromolecular complexes. Ca2+ binding sites usually consist of several acidic residues chelating to bound Ca2+ in a pentagonal bipyramidal manner (2Strynadka N.C.J. James M.N.G. Curr. Opin. Struct. Biol. 1991; 1: 905-914Crossref Scopus (50) Google Scholar). The close locations of acidic residues in a Ca2+ binding site should negatively affect protein stability due to charge repulsion between the acidic residues. How does the introduction of charged residues within a calcium binding site affect the stability and Ca2+ binding behavior? There are some reports describing the effect of mutations within Ca2+ binding sites on the stability of the protein (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar, 4Haezebrouck P. De Baetselier A. Joniau M. Van Deal H. Rosenberg S. Hanssens I. Protein Eng. 1993; 6: 643-649Crossref PubMed Scopus (20) Google Scholar). Our approach in this paper is to introduce the minimum perturbation by amino acid replacement and determine the effect of these mutations on the stability and the Ca2+ binding function using calorimetry. Furthermore, these effects are interpreted in terms of the structural information from x-ray crystallography. For this purpose, we chose human lysozyme because lysozymes are known to be a suitable model for studying protein function and stability (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar, 5Kuroki R. Taniyama Y. Seko C. Nakamura H. Kikuchi M. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6903-6907Crossref PubMed Scopus (104) Google Scholar, 6Kuroki R. Inaka K. Taniyama Y. Kidokoro S. Matsushima M. Kikuchi M. Yutani K. Biochemistry. 1992; 31: 8323-8328Crossref PubMed Scopus (72) Google Scholar, 7Kuroki R. Nitta K. Yutani K. J. Biol. Chem. 1992; 267: 24297-24301Abstract Full Text PDF PubMed Google Scholar). More than 90 sequences of chicken type lysozymes and α-lactalbumins are in the sequence data base. Among these lysozymes, several have been found to have Ca2+ binding ability (8Nitta K. Tsuge H. Sugai S. Shimazaki K. FEBS Lett. 1987; 223: 405-408Crossref PubMed Scopus (66) Google Scholar, 9Nitta K. Tsuge H. Shimazaki K. Sugai S. Biol. Chem. Hoppe-Seyler. 1988; 369: 671-675Crossref PubMed Scopus (51) Google Scholar, 10Godovac-Zimmerman J. Conti A. Napolitano L. Biol. Chem. Hoppe-Seyler. 1988; 369: 1109-1115Crossref PubMed Scopus (37) Google Scholar, 11Jollès P. Lysozymes: Model Enzymes in Biochemistry and Biology. Birkhäuser Verlag, Basel1996Crossref Google Scholar). Three residues (Ala-83, Gln-86, and Ala-92) in human lysozyme are usually mutated to Lys, Asp, and Asp, respectively, in natural Ca2+ binding lysozymes and α-lactalbumins as shown in Fig.1. We have already found that only the double mutation (Gln-86 → Asp and Ala-92 → Asp) in human lysozyme resulted in the formation of a Ca2+ binding site (5Kuroki R. Taniyama Y. Seko C. Nakamura H. Kikuchi M. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6903-6907Crossref PubMed Scopus (104) Google Scholar). The precise analyses of the stability (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar, 5Kuroki R. Taniyama Y. Seko C. Nakamura H. Kikuchi M. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6903-6907Crossref PubMed Scopus (104) Google Scholar, 6Kuroki R. Inaka K. Taniyama Y. Kidokoro S. Matsushima M. Kikuchi M. Yutani K. Biochemistry. 1992; 31: 8323-8328Crossref PubMed Scopus (72) Google Scholar) and the Ca2+affinity (7Kuroki R. Nitta K. Yutani K. J. Biol. Chem. 1992; 267: 24297-24301Abstract Full Text PDF PubMed Google Scholar) using calorimetry have also been performed, and the high resolution structural data of the wild type (12Artymiuk P.J. Blake C.C.F. J. Mol. Biol. 1981; 152: 737-762Crossref PubMed Scopus (263) Google Scholar) and Ca2+binding mutant (13Inaka K. Kuroki R. Kikuchi M. Matsushima M. J. Biol. Chem. 1991; 266: 20666-20671Abstract Full Text PDF PubMed Google Scholar) lysozymes from x-ray crystallography are available. Here we show the effect of the subsequent mutations (Gln-86 → Asp, Ala-92 → Asp, and Ala-83 → Lys) on the Ca2+ binding properties, conformational stabilities, and tertiary structures of these mutants. It was found that both aspartic acids (Asp-86 and Asp-86) are essential for Ca2+ binding. The introduction of an aspartic acid at the Ala-92 position resulted in destabilization of lysozyme, whereas the introduction of an aspartic acid at the Gln-86 position or the introduction of positive charge the Ala-83 position did not affect the stability. The observed instability with the Ala-92 → Asp mutation is proposed to result from the difference in the hydration acidic residues accompanying some basic residues are characteristically associated to make a Ca2+ binding site in Ca2+ binding proteins. Acidic residues are found to directly chelate to Ca2+ in a pentagonal bipyramidal manner (2Strynadka N.C.J. James M.N.G. Curr. Opin. Struct. Biol. 1991; 1: 905-914Crossref Scopus (50) Google Scholar, 13Inaka K. Kuroki R. Kikuchi M. Matsushima M. J. Biol. Chem. 1991; 266: 20666-20671Abstract Full Text PDF PubMed Google Scholar), and some basic residues located in the vicinity of Ca2+ binding sites appear to provide a counter ion charge to the chelating residues. Three chelating aspartic acids, Asp-86, Asp-91, and Asp-92 and the positively charged Lys-83 are conserved in Ca2+ binding lysozymes and α-lactalbumins. Because Asp-91 already exists in wild type human lysozyme (Fig. 1), the effect of the three mutations, Gln-86 → Asp, Ala-92 → Asp, and Ala-83 → Lys on the stability and affinity of Ca2+ was investigated using calorimetry. In Table V, the effect of the mutations on the stability is summarized, in which ΔΔG at 80 °C was calculated according to Becktel and Shellman (27Becktel W.J. Schellman J.A. Biopolymers. 1987; 26: 1859-1877Crossref PubMed Scopus (940) Google Scholar) by assuming that ΔS and ΔC p values of Ca2+ bound and unbound mutants are the same as those of the holo-Q86D/A92D and the wild type lysozymes, respectively, as reported previously (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar). In the presence of 10 mm Ca2+, the mutant A83K/Q86D/A92D showed about 3.6 kcal/mol stabilization, which is similar to that of Q86D/A92D lysozyme. Other mutants, and lysozymes, did not show in the presence of to Schellman J.A. Biopolymers. Scopus Google Scholar, J.A. Biopolymers. Scopus Google Scholar), the of binding such as a Ca2+ binding should the stability of the protein in the presence of the of 10 which is about in of the protein should result in of the In both and mutant lysozymes, was observed in the presence of Ca2+ the mutants Q86D/A92D and that both the and mutants not have Ca2+ affinity, which is with the of the binding as in Table also that both acidic residues Asp-86 and Asp-92 are essential for Ca2+ binding in the mutant human lysozyme. In the of Ca2+, we the effect of the mutation on the stability of lysozyme. The stability of lysozyme was the same as that of the wild type lysozyme, indicating affect of the mutation from Gln-86 to The lysozyme was about 1.3 kcal/mol less than the wild which is similar to the and lysozymes, indicating that the mutation Ala-83 to Lys did not affect the stability the mutation Ala-92 to Asp resulted in 1.3 kcal/mol for the of the mutant human lysozymes and values were to 80 The ΔS and ΔC p for Ca2+ bound mutants in 10 mm were to be the same as those of the and the ΔS and ΔC p for Ca2+ unbound mutants and were to be the same as those of the wild type lysozyme reported reported reported (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google ΔΔG values were to 80 The ΔS and ΔC p for Ca2+ bound mutants in 10 mm were to be the same as those of the (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar), and the ΔS and ΔC p for Ca2+ unbound mutants and were to be the same as those of the wild type lysozyme (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar). in a the stability in terms of the structural high resolution structural data were from x-ray crystallography. these aspartic side chains are located within to chelate Ca2+ and lysozymes in and the introduction of aspartic acid at position resulted in a of the side the of the site as if to reduce the charge repulsion to Asp-91 lysozyme in The charged of Asp-86, and Lys-83 are located and 10 from Asp-91 in the wild type lysozyme, the mutations Gln-86 → Asp or Ala-83 → Lys did not affect the stability. It that a charged a Ca2+ binding site does not the protein stability. a of the side be to reduce the repulsion between the because of the It been reported that an ion located on the of a protein does not affect the stability of the protein U. H. Biochemistry. 1991; PubMed Scopus Google Scholar). the a destabilization was observed for the mutation Ala-92 → for this destabilization is the difference in hydration between the and Asp side chains The tertiary of the mutant that the side of Asp-92 is and the side was similar to those of the holo-Q86D/A92D lysozyme to and M. J. Biochemistry. 1989; Scopus Google Scholar, M. Mol. Biol. 1993; PubMed Scopus Google Scholar), the difference in hydration between and is calculated to be about kcal/mol at 80 Because Asp-92 in the mutant and Ala-92 in the wild type structures are with the of these amino acids calculated to be less than the difference in hydration is considered to be the part of the instability kcal/mol in Table observed in the mutants The than effect on stability be by the binding of ion to Asp-92 in the as in the crystal structures of the and lysozyme. acidic residues accompanying some basic residues are characteristically associated to make a Ca2+ binding site in Ca2+ binding proteins. Acidic residues are found to directly chelate to Ca2+ in a pentagonal bipyramidal manner (2Strynadka N.C.J. James M.N.G. Curr. Opin. Struct. Biol. 1991; 1: 905-914Crossref Scopus (50) Google Scholar, 13Inaka K. Kuroki R. Kikuchi M. Matsushima M. J. Biol. Chem. 1991; 266: 20666-20671Abstract Full Text PDF PubMed Google Scholar), and some basic residues located in the vicinity of Ca2+ binding sites appear to provide a counter ion charge to the chelating residues. Three chelating aspartic acids, Asp-86, Asp-91, and Asp-92 and the positively charged Lys-83 are conserved in Ca2+ binding lysozymes and α-lactalbumins. Because Asp-91 already exists in wild type human lysozyme (Fig. 1), the effect of the three mutations, Gln-86 → Asp, Ala-92 → Asp, and Ala-83 → Lys on the stability and affinity of Ca2+ was investigated using calorimetry. In Table V, the effect of the mutations on the stability is summarized, in which ΔΔG at 80 °C was calculated according to Becktel and Shellman (27Becktel W.J. Schellman J.A. Biopolymers. 1987; 26: 1859-1877Crossref PubMed Scopus (940) Google Scholar) by assuming that ΔS and ΔC p values of Ca2+ bound and unbound mutants are the same as those of the holo-Q86D/A92D and the wild type lysozymes, respectively, as reported previously (3Kuroki R. Kawakita H. Nakamura H. Yutani K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6803-6807Crossref PubMed Scopus (59) Google Scholar). In the presence of 10 mm Ca2+, the mutant A83K/Q86D/A92D showed about 3.6 kcal/mol stabilization, which is similar to that of Q86D/A92D lysozyme. Other mutants, and lysozymes, did not show in the presence of to Schellman J.A. Biopolymers. Scopus Google Scholar, J.A. Biopolymers. Scopus Google Scholar), the of binding such as a Ca2+ binding should the stability of the protein in the presence of the of 10 which is about in of the protein should result in of the In both and mutant lysozymes, was observed in the presence of Ca2+ the mutants Q86D/A92D and that both the and mutants not have Ca2+ affinity, which is with the of the binding as in Table also that both acidic residues Asp-86 and Asp-92 are essential for Ca2+ binding in the mutant human lysozyme. In the of Ca2+, we the effect of the mutation on the stability of lysozyme. The stability of lysozyme was the same as that of the wild type lysozyme, indicating affect of the mutation from Gln-86 to The lysozyme was about 1.3 kcal/mol less than the wild which is similar to the and lysozymes, indicating that the mutation Ala-83 to Lys did not affect the stability the mutation Ala-92 to Asp resulted in 1.3 kcal/mol the stability in terms of the structural high resolution structural data were from x-ray crystallography. these aspartic side chains are located within to chelate Ca2+ and lysozymes in and the introduction of aspartic acid at position resulted in a of the side the of the site as if to reduce the charge repulsion to Asp-91 lysozyme in The charged of Asp-86, and Lys-83 are located and 10 from Asp-91 in the wild type lysozyme, the mutations Gln-86 → Asp or Ala-83 → Lys did not affect the stability. It that a charged a Ca2+ binding site does not the protein stability. a of the side be to reduce the repulsion between the because of the It been reported that an ion located on the of a protein does not affect the stability of the protein U. H. Biochemistry. 1991; PubMed Scopus Google Scholar). the a destabilization was observed for the mutation Ala-92 → for this destabilization is the difference in hydration between the and Asp side chains The tertiary of the mutant that the side of Asp-92 is and the side was similar to those of the holo-Q86D/A92D lysozyme to and M. J. Biochemistry. 1989; Scopus Google Scholar, M. Mol. Biol. 1993; PubMed Scopus Google Scholar), the difference in hydration between and is calculated to be about kcal/mol at 80 Because Asp-92 in the mutant and Ala-92 in the wild type structures are with the of these amino acids calculated to be less than the difference in hydration is considered to be the part of the instability kcal/mol in Table observed in the mutants The than effect on stability be by the binding of ion to Asp-92 in the as in the crystal structures of the and lysozyme. We to M. Kikuchi and for mutant lysozymes, and for of human lysozyme. We also L. H. and of of for data of mutant We to and for and of the