Utility of 5-Cyanotryptophan Fluorescence as a Sensitive Probe of Protein Hydration.
Utility of 5-Cyanotryptophan Fluorescence as a Sensitive Probe of Protein Hydration.
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DOI：
10.1021/acs.jpcb.5b12233
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发表时间：
2016-02-11
期刊：
影响因子：
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通讯作者：
Gai F
中科院分区：
文献类型：
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作者：
Markiewicz BN;Mukherjee D;Troxler T;Gai F
色氨酸（Trp）荧光已被广泛用于研究蛋白质的结构、动力学和功能。特别是，它提供了一种方便和特定位点的方法来探测蛋白质的水合状态和动力学。在这里，我们发现色氨酸类似物5-氰色氨酸（TrpCN）也可以用于此目的，但具有增强水合敏感性的好处。这一结论是基于对5-氰吲哚、TrpCN和含TrpCN的多肽在不同溶剂中的静态和时间分辨荧光性质的测量得出的，这表明在脱水后，TrpCN的荧光量子产率（QY）和寿命（τF）的变化要比Trp大得多。例如，在水中TrpCN的QY小于0.01，在1,4-二氧六烷中增加到0.11。TrpCN在水中的荧光衰减动力学由0.4 ns组分主导，而在1,4-二氧六环中，荧光衰减动力学由6.0 ns组分主导。TrpCN作为一个敏感的荧光报告者的多功能用途在三个应用中得到进一步证明，我们使用它(1)探测由二甲亚砜和H2O组成的二元混合物的溶剂性质，(2)监测抗菌肽与脂质膜的结合相互作用，(3)在折叠蛋白质中区分两种不同的水合环境。
Tryptophan (Trp) fluorescence has been widely used to interrogate the structure, dynamics and function of proteins. In particular, it provides a convenient and site-specific means to probe a protein’s hydration status and dynamics. Herein, we show that a tryptophan analog, 5-cyanotryptophan (TrpCN), can also be used for this purpose, but with the benefit of enhanced sensitivity to hydration. This conclusion is reached based on measurements of the static and time-resolved fluorescence properties of 5-cyanoindole, TrpCN, and TrpCN–containing peptides in different solvents, which indicate that upon dehydration the fluorescence quantum yield (QY) and lifetime (τF) of TrpCN undergo a much greater change in comparison to those of Trp. For example, in H2O the QY of TrpCN is less than 0.01, which increases to 0.11 in 1,4-dioxane. Consistently, the fluorescence decay kinetics of TrpCN in H2O are dominated by a 0.4 ns component, whereas in 1,4-dioxane the kinetics are dominated by a 6.0 ns component. The versatile utility of TrpCN as a sensitive fluorescence reporter is further demonstrated in three applications, where we used it (1) to probe the solvent property of a binary mixture consisting of dimethyl sulfoxide and H2O, (2) to monitor the binding interaction of an antimicrobial peptide with lipid membranes, and (3) to differentiate two differently hydrated environments in a folded protein.