Enhancement of the Thermostability of Hydrogenobacter thermophilus Cytochrome c552 through Introduction of an Extra Methylene Group into Its Hydrophobic Protein Interior

详细信息    查看全文

• 作者：Hulin Tai ; Kiyofumi Irie ; Shin-ichi Mikami ; Yasuhiko Yamamoto
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：April 19, 2011
• 年：2011
• 卷：50
• 期：15
• 页码：3161-3169
• 全文大小：880K
• 年卷期：v.50,no.15(April 19, 2011)
• ISSN：1520-4995

文摘

Careful scrutiny of the protein interior of Hydrogenobacter thermophilus cytochrome c₅₅₂ (HT) on the basis of its X-ray structure [Travaglini-Allocatelli, C., Gianni, S., Dubey, V. K., Borgia, A., Di Matteo, A., Bonivento, D., Cutruzzola, F., Bren, K. L., and Brunori, M. (2005) J. Biol. Chem. 280, 25729鈭?5734] indicated that a void space, which is large enough to accommodate a methyl group, exists in the hydrophobic protein interior near the heme. We tried to reduce the void space through the replacement of a Val by Ile or Leu (Val/Ile or Val/Leu mutation), and then the structural and functional consequences of these two mutations were characterized in order to elucidate the relationship between the nature of the packing of hydrophobic residues and the functional properties of the protein. The study demonstrated striking differences in the structural and functional consequences between the two mutations. The Val/Ile mutation was found to cause further enhancement of the thermostability of the oxidized HT, as reflected in the increase of the denaturation temperature (T_m) value by 3 deg, whereas the thermostability of the reduced form was essentially unaffected. As a result, the redox potential (E_m) of the Val/Ile mutant exhibited a negative shift of 50 mV relative to that of the wild-type protein in an enthalpic manner, this being consistent with our previous finding that a protein with higher stability in its oxidized form exhibits a lower E_m value [Terui, N., Tachiiri, N., Matsuo, H., Hasegawa, J., Uchiyama, S., Kobayashi, Y., Igarashi, Y., Sambongi, Y., and Yamamoto, Y. (2003) J. Am. Chem. Soc. 125, 13650鈭?3651]. In contrast, the Val/Leu mutation led to a decrease in thermostability of both the redox forms of the protein, as reflected in the decreases of the T_m values of the oxidized and reduced proteins by 3 and 5 deg, respectively, and the E_m value of the Val/Leu mutant happened to be similar to that of the Val/Ile one. The E_m value of the Val/Leu mutant could be reasonably interpreted in terms of the different effects of the mutation on the stabilities of the two different redox forms of the protein. Thus, the present study demonstrated that the stability of the protein is affected quite sensitively by the contextual stereochemical packing of hydrophobic residues in the protein interior and that the structural properties of the hydrophobic core in the protein interior are crucial for control of the redox function of the protein. These findings provide novel insights as to functional control of a protein, which could be utilized for tuning of the T_m and E_m values of the protein by means of protein engineering.