Halide and proton binding kinetics of yellow fluorescent protein variants.
Halide and proton binding kinetics of yellow fluorescent protein variants.
复制标题
黄色荧光蛋白变体的卤化物和质子结合动力学。
DOI：
10.1021/bi3016839
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
Clive R. Bagshaw
中科院分区：
文献类型：
--
作者：
H. E. Seward;J. Basran;Roanne Denton;M. Pfuhl;F. Muskett;Clive R. Bagshaw
绿色荧光蛋白中的T203 Y取代导致发射红移，产生一类称为黄色荧光蛋白（YFP）的突变体。这些YFP突变体中的许多以毫摩尔范围内的亲和力结合卤化物，这通常导致发色团pK值偏移到生理范围内。虽然这种敏感性可以用于卤化物和pH传感器，但在其他研究中，如在Förster共振能量转移探针中测量融合蛋白内的构象变化时，需要降低这种环境敏感性。金星和黄水晶是两个这样的变种，已开发出大大降低卤化物的敏感性。在这里，我们比较了几种YFP变体的卤化物结合动力学和偶联质子化反应，并检测到缓慢的动力学（解离速率常数在0.1-1 s（-1）的范围内），表明在所有情况下与内部位点结合。与原始YFP 10 C构建体相比，Venus和Citrine的有效卤化物亲和力大大降低，主要是通过降低的缔合速率常数。YFP 10 C的核磁共振研究证实，卤化物结合发生在缓慢的时间尺度上（<4 s（-1）），并且化学位移的扰动发生在整个序列和结构中。
A T203Y substitution in green fluorescent protein causes a red shift in emission to yield a class of mutants known as yellow fluorescent protein (YFP). Many of these YFP mutants bind halides with affinities in the millimolar range, which often results in the chromophore pK values being shifted into the physiological range. While such sensitivities may be exploited for halide and pH sensors, it is desirable to reduce such environmental sensitivities in other studies, such as in Förster resonance energy transfer probes to measure conformational changes within fusion proteins. Venus and Citrine are two such variants that have been developed with much reduced halide sensitivities. Here we compare the kinetics of halide binding, and the coupled protonation reaction, for several YFP variants and detect slow kinetics (dissociation rate constants in the range of 0.1-1 s(-1)), indicative of binding to an internal site, in all cases. The effective halide affinity for Venus and Citrine is much reduced compared with that of the original YFP 10C construct, primarily through a reduced association rate constant. Nuclear magnetic resonance studies of YFP 10C confirm halide binding occurs on a slow time scale (<4 s(-1)) and that perturbations in the chemical shift occur throughout the sequence and structure.
影响因子：
5.6
作者：
R. Wachter;D. Yarbrough;K. Kallio;S. Remington
通讯作者：
R. Wachter;D. Yarbrough;K. Kallio;S. Remington