Protein-engineering study of contribution of conceivable D-serine residues to the thermostabilization of ovalbumin under alkaline conditions.
During storage of shell eggs, an egg-white protein, ovalbumin is converted into a molecular species with higher thermostability (Delta T(m)=8 degrees), which is named as S-ovalbumin. Since the pH of egg white is raised to 9.5 in that process, particular chemical modifications could occur. Although any evidence for relationship between chemical modifications and the thermostabilization had not been established, our X-ray crystallographic model for S-ovalbumin demonstrated that three serine residues (S164, S236, and S320) might be in D-configuration. Then, whether these serine residues contribute to the thermostabilization in S-ovalbumin is investigated by protein-engineering approach. By alkaline treatment, S236G mutant was converted to a more thermostable form, as well as ovalbumin has been converted to S-ovalbumin. Hence, the Ser236 is regarded to have no contribution to S-ovalbumin formation. On the contrary, S164G and S320G did show accelerated conversion to thermostable forms in comparison with ovalbumin or wild-type recombinant protein. When these residues were replaced with valine, which has a slower racemization rate, Delta T(m) after alkaline treatment had become half of that for the wild-type recombinant ovalbumin. Therefore, the process of S-ovalbumin formation is deduced to consist of two steps, which is closely related to the two serine residues.
Takahashi
Nobuyuki
N
Department of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan. nobi@kais.kyoto-u.ac.jp
Maeda
Maki
M
Yamasaki
Masayuki
M
Mikami
Bunzo
B
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010047:Ovalbumin / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry*; D013696:Temperature
C104026:S-ovalbumin; D012694:Serine; D010047:Ovalbumin