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首页> 外文期刊>Biochemistry >Enhancement of the Thermostability of Hydrogenobacter thermophilus Cytochrome c_(552) through Introduction of an Extra Methylene Group into Its Hydrophobic Protein Interior
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Enhancement of the Thermostability of Hydrogenobacter thermophilus Cytochrome c_(552) through Introduction of an Extra Methylene Group into Its Hydrophobic Protein Interior

机译：通过将额外亚甲基引入其疏水蛋白内部，增强氢杆菌CO（552）的热稳定性

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摘要

Careful scrutiny of the protein interior of Hydrogenobacter thermophilus cytochrome c_(552) (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, 25729-25734] 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, 13650-13651]. 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.
机译：仔细仔细仔细审查氢杆菌硫杆菌细胞色素C_（552）（HT）的蛋白质内部[Travaglini-Allocatelli，C.，Gianni，S.，Dubey，VK，Borgia，A.，Di Matteo ，A.，Bonivento，D.，Cutruzzola，F.，Bren，KL和Brunori，M。（2005）J.Biol。化学。 280,25729-25734]表明，空隙空间足以容纳甲基，在血红素附近的疏水蛋白质内部存在。我们试图通过ILE或LEU（VAL / ILE或VAL / LEU突变）替换VAL来减少空隙空间，然后表征了这两个突变的结构和功能后果，以阐明性质之间的关系疏水残基的包装及蛋白质的功能性质。该研究表明，两种突变之间的结构和功能后果的显着差异。发现VAL / ILE突变导致氧化HT的热稳定性进一步提高了氧化HT的热稳定性，如在变性温度（T_M）值的增加中〜3°的增加，而还原形式的热稳定性基本上不受影响。结果，VAL / ILE突变体的氧化还原电位（E_M）以焓的方式表现出〜50mV的负偏移，这与我们之前发现具有更高的蛋白质的蛋白质一致其氧化形式的稳定性表现出较低的E_M值[Terui，N.，Tachiiri，N.，Matsuo，H.，Haegawa，J。，Uchiyama，S.，Kobayashi，Y.，Igarashi，Y.，Sambongi，Y. ，yamamoto，y。（2003）J.AM。化学。 SOC。 125,13650-13651]。相反，Val / Leu突变导致蛋白质的氧化还原形式的热稳定性降低，如分别氧化和减少蛋白质的T_M值的降低，分别由〜3和〜5°的减少。 val / leu突变体的E_M值恰好与Val / ILE I类似。 Val / Leu突变体的E-M值可以在突变对蛋白质两种不同氧化还原形式的稳定性的不同效果方面合理地解释。因此，本研究表明，通过蛋白质内部的疏水残留物的疏水残留物的上下文立体化学包​​装，蛋白质的稳定性敏感地受到敏感的影响，并且蛋白质内部的疏水芯的结构性质对于控制氧化还原功能至关重要蛋白质。这些发现提供了对蛋白质的功能控制的新颖见解，其可用于通过蛋白质工程调节蛋白质的T_M和E_M值。

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  • 来源
    《Biochemistry》 |2011年第15期|共9页
  • 作者

    Tai HL; Irie K; Mikami S; Yamamoto Y;

  • 作者单位

    Department of Chemistry University of Tsukuba Tsukuba 305-8571 Japan;

    Department of Chemistry University of Tsukuba Tsukuba 305-8571 Japan;

    Department of Chemistry University of Tsukuba Tsukuba 305-8571 Japan;

    Department of Chemistry University of Tsukuba Tsukuba 305-8571 Japan;

  • 收录信息
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    Enhancement; Thermostability; Introduction;

    机译：增强;热稳定性;介绍;

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