Novel chromophores and buried charges control color in mFruits
Novel chromophores and buried charges control color in mFruits
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DOI：
10.1021/bi060773l
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发表时间：
2006-08-15
期刊：
影响因子：
2.9
通讯作者：
Remington, S. James
中科院分区：
文献类型：
--
作者：
Shu, Xiaokun;Shaner, Nathan C.;Remington, S. James
mFruits是第二代单体红色荧光蛋白（mRFPs），与第一代mRFP1相比，它们具有更高的亮度和光稳定性。发射最大值和激发最大值分别分布在550 ~ 650 nm和540 ~ 590nm的较大范围内；然而，光谱的变化可以追溯到几个关键的氨基酸。对三种代表物质，mOrange， mStrawberry和mCherry的光谱和原子分辨率晶体学分析揭示了不同的机制来建立激发和发射最大值。显然，它们都经过第二氧化步骤，在多肽主链上产生一个酰基胺键。与前体dred相比，直接共价修饰该连锁（mOrange）和间接修饰发色团环境（mStrawberry和mCherry）产生强烈的蓝移和红移变体。mOrange的蓝移是由蛋白质骨架的共价修饰引起的。电子密度图表明，在Thr 66 O γ与多肽主链反应后，形成了第三个杂环，2-羟基-二氢恶唑，这反过来又减少了65位羰基与其他发色团的结合。在mStrawberry和mCherry中，带电lys70的移动和Glu 215的质子化改变了发色团的电子密度分布，引起了红移。mCherry和mStrawberry的pH依赖谱移似乎是由Glu 215的滴定引起的，尽管对于mStrawberry来说，Thr 66的部分环化可能在高pH下起作用。
mFruits are second-generation monomeric red fluorescent proteins (mRFPs) that have improved brightness and photostability compared to the first-generation mRFP1. The emission and excitation maxima are distributed over the remarkably large ranges of about 550-650 and 540-590 nm, respectively; however, the variations in the spectra can be traced to a few key amino acids. Spectroscopic and atomic resolution crystallographic analyses of three representatives, mOrange, mStrawberry, and mCherry, reveal that different mechanisms operate to establish the excitation and emission maxima. Evidently, they all undergo the second oxidation step to produce an acylimine linkage in the polypeptide backbone. In comparison to the progenitor DsRed, direct covalent modification to this linkage ( mOrange) and indirect modification of the chromophore environment ( mStrawberry and mCherry) produce strong blue- and red-shifted variants. The blue shift of mOrange is induced by an unprecedented covalent modification of the protein backbone. The electron-density map indicates the formation of a third heterocycle, 2-hydroxy-dihydrooxazole, upon the reaction of Thr 66 O gamma with the polypeptide backbone, which in turn reduces the conjugation of the carbonyl at position 65 with the rest of the chromophore. In mStrawberry and mCherry, the movement of charged Lys 70 and protonation of Glu 215 are proposed to modify the chromophore electron-density distribution, inducing the red shift. pH-dependent spectral shifts of mCherry and mStrawberry appear to result from the titration of Glu 215, although, for mStrawberry, partial cyclization of Thr 66 may contribute at high pH.