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mCLIFY 的亮度的特征和结构基础:一种新的单体和循环 permuted 亮黄色光蛋白质

Him Shweta^1,2,3,4, Kushol Gupta⁵, Yufeng Zhou³

¹Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA-19104, United States of America.

Research square

|July 29, 2024

概括

我们开发了mCLIFY,一种明亮,黄色的光蛋白 (FP),非常适合用于细胞和单分子研究. 这种单体FP克服了YPet.

科学领域:

生物化学生物化学
生物物理学的生物物理.
分子生物学分子生物学

背景情况:

YPet是一种明亮黄色的光蛋白 (FP),广泛用于细胞和单分子研究.
由于YPet的二元化倾向,可能会导致由于homo-Förster共振能量转移 (homo-FRET) 的定量错误.
对于先进的生物成像,需要单质,明亮和光稳定的光蛋白.

研究的目的:

设计基于YPet.Pet的单体,明亮和长寿命的黄色光蛋白 (FP).
描述工程FP的光物理和结构性质,命名为mCLIFY.
阐明YPet二元化和mCLIFY增强性能的结构基础.

主要方法:

循环 permutation 的 YPet,以创建单体的 mCLIFY.
使用X射线晶体学 (1.57 Å分辨率) 确定mCLIFY的原子结构.
光物理特征,包括量子产量,灭绝系数和寿命测量.
使用YPet和mCLIFY进行二元化研究,确定解离常数 (KD).

主要成果:

mCLIFY成功地被设计成一种单体,明亮,黄色和寿命长的FP.
由于V58L突变,mCLIFY表现出增强的光物理特性,比金星亮30%左右.
发现YPet以3.4μM的KD进行二元化,导致homo-FRET和定量错误.
mCLIFY 的晶体结构为其增强的亮度和单性质提供了洞察力.

结论:

mCLIFY是YPet的优质替代品,用于需要单体黄色FP的应用,最大限度地减少二元化工件.
V58L突变显著提高了mCLIFY的亮度和量子产量.
了解FP二分化和光物理学的结构基础,可以合理设计改进的光探针.

关键词:

光蛋白质是一种光蛋白质.在X射线晶体学.分析超离心法分析超离心法循环变换的循环变换是一种循环变换.光的寿命光的寿命光共振能量转移的能量转移.小角X射线散射的小角度X射线散射.

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