Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness
Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness
复制标题
mCherry 变体的构象动力学：侧链运动与荧光亮度之间的联系
DOI：
10.1021/acs.jpcb.2c05584
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Jimenez, Ralph
中科院分区：
文献类型：
--
作者：
Mukherjee, Srijit;Manna, Premashis;Douglas, Nancy;Chapagain, Prem P.;Jimenez, Ralph
最近开发的mCherry-XL红色荧光蛋白(FP)的亮度比其祖先mCherry高3倍，这是因为其荧光量子产率增加的基础是非辐射衰减率的显著降低。为了研究区分两个FP和密切相关变体的四个突变的结构和动力学作用，我们使用了微秒时间尺度的全原子分子动力学模拟。模拟表明，I197R突变导致多个氢键接触的形成，并增加了β枪管的刚性。特别是，mCherryXL显示β7和β10-链之间的间隙的纳秒时间尺度呼吸减少，这是以前被证明是mCherry最灵活的区域。结合实验结果，模拟还揭示了残基161的空间相互作用以及生色团与59、143和163位残基的氢键相互作用网络，这对扰动生色团的电子结构是关键的。最后，我们阐明了保守残基R95和S146的构象动力学，它们与发色团氢键相连，并为观察到的光物理提供了物理见解。据我们所知，这是第一个评估定向进化产生的一组密切相关FP的构象空间的研究。
The 3-fold higher brightness of the recently developed mCherry-XL red fluorescent protein (FP) compared to its progenitor, mCherry, is due to a significant decrease in the nonradiative decay rate underlying its increased fluorescence quantum yield. To examine the structural and dynamic role of the four mutations that distinguish the two FPs and closely related variants, we employed microsecond time scale, all-atom molecular dynamics simulations. The simulations revealed that the I197R mutation leads to the formation of multiple hydrogen-bonded contacts and increased rigidity of the β-barrel. In particular, mCherryXL showed reduced nanosecond time scale breathing of the gap between the β7 and β10-strands, which was previously shown to be the most flexible region of mCherry. Together with experimental results, the simulations also reveal steric interactions of residue 161 and a network of hydrogen-bonding interactions of the chromophore with residues at positions 59, 143, and 163 that are critical in perturbing the chromophore electronic structure. Finally, we shed light on the conformational dynamics of the conserved residues R95 and S146, which are hydrogen-bonded to the chromophore, and provide physical insights into the observed photophysics. To the best of our knowledge, this is the first study that evaluates the conformational space for a set of closely related FPs generated by directed evolution.