1LAW

STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY CAVITY-FILLING MUTATIONS WITHIN A HYDROPHOBIC CORE

1LAW の概要

• エントリーDOI: 10.2210/pdb1law/pdb
• 分子名称: RIBONUCLEASE H (2 entities in total)
• 機能のキーワード: hydrolase
• 由来する生物種: Escherichia coli
• 細胞内の位置: Cytoplasm : P0A7Y4
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 17637.03

構造登録者

Ishikawa, K.,Nakamura, H.,Morikawa, K.,Kanaya, S. (登録日: 1993-05-10, 公開日: 1993-10-31, 最終更新日: 2024-02-14)

主引用文献

Ishikawa, K.,Nakamura, H.,Morikawa, K.,Kanaya, S.
Stabilization of Escherichia coli ribonuclease HI by cavity-filling mutations within a hydrophobic core.
Biochemistry, 32:6171-6178, 1993

PubMed Abstract: The crystal structure of Escherichia coli ribonuclease HI has a cavity near Val-74 within the protein core. In order to fill the cavity space, we constructed two mutant proteins, V74L and V74I, in which Val-74 was replaced with either Leu or Ile, respectively. The mutant proteins are stabilized, as revealed by a 2.1-3.7 degrees C increase in the Tm values, as compared to the wild-type protein at pH values of 3.0 and 5.5. The mutant protein V74A, in which Val-74 is replaced with Ala, was also constructed to analyze the reverse effect. The stability of V74A decreases by 7.6 degrees C at pH 3.0 and 12.7 degrees C at pH 5.5 in Tm as compared to those values for the wild-type protein. None of the three mutations significantly affect the enzymatic activity. The crystal structures of V74L and V74I, determined at 1.8-A resolution, are almost identical to that of the wild-type protein, except for the mutation site. In the two mutant proteins, calculation by the Voronoi procedure shows that the cavity volumes around the individual mutation sites are remarkably reduced as compared to that in the wild-type protein. These results indicate that the introduction of a methylene group into the cavity, without causing steric clash, contributes to an increase in the hydrophobic interaction within the protein core and thereby enhances protein stability. We also discuss the role of the Leu side chain, which can assume many different local conformations on a helix without sacrificing thermostability.

PubMed: 8390295
DOI: 10.1021/bi00075a009

実験手法

X-RAY DIFFRACTION (1.8 Å)