mKate2-K67R/R197H—Extra-Bright Red Fluorescent Biomarker of New Generation. X-Ray Structure and Molecular Dynamic Properties

IF 16.4 1区化学 Q1 CHEMISTRY, MULTIDISCIPLINARY

Accounts of Chemical Research Pub Date : 2024-08-05 DOI:10.1134/S1068162024040113

E. A. Goryacheva, A. V. Rossokhin, D. A. Ruchkin, A. M. Bogdanov, I. V. Artemyev, N. V. Pletneva, V. Z. Plenev

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引用次数: 0

Abstract

Objective: Cell biology continuously shows the need for new fluorescent tags with advanced properties. The object of our current study is a new genetically encoded monomeric red fluorescent biomarker mKate2-K67R/R197H (λex/λem 579/603 mn), designed from commercial biomarker mKate2 by two R197H/K67R mutations. The mKate2 precursor, a far-red fluorescent protein, is nearly 3-fold brighter than the previously designed mKate. Compared with commercial mKate2, the double mutant mKate2-K67R/R197H (alternative names FusionRed2 and Diogenes) exhibits an additional ~1.6-fold increase in fluorescence brightness and represents the next generation of extra-bright red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals. Methods: The paper presents the results of X-ray and molecular dynamics study of new bright biomarker mKate2-K67R/R197H. Results and Discussion: The three dimensional structure of new advanced red fluorescent biomarker mKate2-K67R/R197H has been studied by X-ray method at 1.5Å resolution supported by molecular dynamics (MD) study The principal structural fold of the protein is an 11-stranded β-barrel. The nearest chromophore environment (≤ 4 Å) comprises 18 tightly packed residues. Conclusions: The MD study showed that the brightness of mKate2-K67R/R197H and its mKate2 precursor correlates with the dipole moments of the amino acid environments of the chromophores. The higher the dipole moment, the higher the brightness of biomarker.

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mKate2-K67R/R197H-新一代超亮红色荧光生物标记物。X 射线结构和分子动力学特性

摘要目的：细胞生物学不断显示出对具有高级特性的新型荧光标记的需求。我们目前的研究对象是一种新的基因编码单体红色荧光生物标记物 mKate2-K67R/R197H（λex/λem 579/603 mn），它是由商业生物标记物 mKate2 通过两个 R197H/K67R 突变设计而成的。mKate2 前体是一种远红荧光蛋白，其亮度是之前设计的 mKate 的近 3 倍。与商用 mKate2 相比，双突变体 mKate2-K67R/R197H（别名 FusionRed2 和 Diogenes）的荧光亮度增加了约 1.6 倍，代表了新一代超亮红色荧光探针，为活细胞和动物体内蛋白质的荧光成像提供了新的可能性。研究方法本文介绍了新型明亮生物标记物 mKate2-K67R/R197H 的 X 射线和分子动力学研究结果。结果与讨论：在分子动力学（MD）研究的支持下，用 1.5 Å 分辨率的 X 射线方法研究了新型高级红色荧光生物标记物 mKate2-K67R/R197H 的三维结构。最近的发色团环境（≤ 4 Å）包括 18 个紧密排列的残基。结论：MD 研究表明，mKate2-K67R/R197H 及其 mKate2 前体的亮度与发色团氨基酸环境的偶极矩相关。偶极矩越大，生物标记物的亮度越高。

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来源期刊

Accounts of Chemical Research 化学-化学综合

CiteScore

31.40

自引率

1.10%

发文量

312

审稿时长

2 months

期刊介绍： Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance. Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.