Contribution of a surface salt bridge to the protein stability of deep-sea Shewanella benthica cytochrome c'

IF 3 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Sotaro Fujii , Riku Sakaguchi , Hiroya Oki , Kazuki Kawahara , Tadayasu Ohkubo , So Fujiyoshi , Yoshihiro Sambongi
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引用次数: 0

Abstract

Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.1 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.

Abstract Image

表面盐桥对深海本氏谢瓦尼拉细胞色素c'蛋白质稳定性的贡献。
深海本氏谢瓦尼拉和紫外谢瓦尼拉的两种同源细胞色素c’,SBCP和SVCP,分别只有9个表面氨基酸取代,N末端有一个。尽管序列差异很小,SBCP在热方面比SVCP更稳定。在这里,我们检查了SBCP变体的热稳定性,每个变体都包含SVCP中九个取代残基中的一个,并发现SBCP K87V变体是最不稳定的。然后,我们以2.1Å的分辨率确定了SBCP K87V变体的X射线晶体结构。该变体保留了类似于野生型的四螺旋束结构,但在突变位点周围的水合结构中观察到显著差异。在野生型中,不是在Lys-87和Asp-91之间形成螺旋内盐桥,而是在Val-87周围通过与附近氨基酸残基的氢键网络观察到包合物状水合作用。这种网络可能会增强周围水分子的有序性,导致蛋白质的熵不稳定。这些结果表明,Val-87周围不利的疏水水合环境和无法形成Asp-91介导的盐桥,导致了SBCP和SVCP之间观察到的稳定性差异。这些发现将在未来的蛋白质工程中有用,通过操纵表面螺旋内盐桥来控制蛋白质的稳定性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of structural biology
Journal of structural biology 生物-生化与分子生物学
CiteScore
6.30
自引率
3.30%
发文量
88
审稿时长
65 days
期刊介绍: Journal of Structural Biology (JSB) has an open access mirror journal, the Journal of Structural Biology: X (JSBX), sharing the same aims and scope, editorial team, submission system and rigorous peer review. Since both journals share the same editorial system, you may submit your manuscript via either journal homepage. You will be prompted during submission (and revision) to choose in which to publish your article. The editors and reviewers are not aware of the choice you made until the article has been published online. JSB and JSBX publish papers dealing with the structural analysis of living material at every level of organization by all methods that lead to an understanding of biological function in terms of molecular and supermolecular structure. Techniques covered include: • Light microscopy including confocal microscopy • All types of electron microscopy • X-ray diffraction • Nuclear magnetic resonance • Scanning force microscopy, scanning probe microscopy, and tunneling microscopy • Digital image processing • Computational insights into structure
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