A Non-perturbing Probe of Coiled Coil Formation Based on Electron Transfer Mediated Fluorescence Quenching
A Non-perturbing Probe of Coiled Coil Formation Based on Electron Transfer Mediated Fluorescence Quenching
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基于电子转移介导荧光猝灭的卷绕线圈形成非扰动探针
DOI：
10.1021/acs.biochem.6b00270
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发表时间：
2016
期刊：
影响因子：
2.9
通讯作者：
Raleigh, Daniel P.
中科院分区：
文献类型：
--
作者：
Watson, Matthew D.;Peran, Ivan;Raleigh, Daniel P.
卷曲螺旋在自然界中是丰富的，在测序的基因组中存在于103%的蛋白质中，并且在从转录因子到结构蛋白的蛋白质中发现。基序仍然是理解蛋白质-蛋白质相互作用的重要模型系统，并且在生物启发材料和合成生物学中的应用越来越多。自组装的热力学知识，特别是解离常数KD，对于设计的卷曲螺旋的应用和理解天然卷曲螺旋的体内特异性是必不可少的。测量KD的标准方法通常依赖于浓度依赖性圆二色性（CD）。荧光方法是一种有吸引力的替代方法;然而，Trp很少在卷曲螺旋的内部位置发现，并且附加非天然荧光团会干扰系统。我们展示了一个简单的，非扰动的方法来监测卷曲螺旋的形成usingp-cyanophenylalanine（FCN）和硒蛋氨酸（MSe），硒类似物的蛋氨酸。FCN荧光可以被选择性地激发，并通过与MSe的电子转移而被有效地猝灭。FCN和MSe都代表卷曲螺旋中的最小扰动取代。FCNfluorescence的MSequenching被证明提供了一种非扰动的方法，用于以下卷曲螺旋的形成和准确地确定解离常数。使用设计的异二聚体卷曲螺旋验证该方法。荧光监测滴定法推导的KD与浓度依赖性CD测量推导的值在测量不确定度范围内非常一致。然而，荧光方法需要较少的蛋白质，耗时较少，可以应用于较低的浓度，并可以应用于高通量筛选。
Coiled coils are abundant in nature, occurring in ∼3% of proteins across sequenced genomes, and are found in proteins ranging from transcription factors to structural proteins. The motif continues to be an important model system for understanding protein–protein interactions and is finding increased use in bioinspired materials and synthetic biology. Knowledge of the thermodynamics of self-assembly, particularly the dissociation constantKD, is essential for the application of designed coiled coils and for understanding thein vivospecificity of natural coiled coils. Standard methods for measuringKDtypically rely on concentration dependent circular dichroism (CD). Fluorescence methods are an attractive alternative; however Trp is rarely found in an interior position of a coiled coil, and appending unnatural fluorophores can perturb the system. We demonstrate a simple, non-perturbing method to monitor coiled coil formation usingp-cyanophenylalanine (FCN) and selenomethionine (MSe), the Se analogue of Met. FCNfluorescence can be selectively excited and is effectively quenched by electron transfer with MSe. Both FCNand MSerepresent minimally perturbing substitutions in coiled coils. MSequenching of FCNfluorescence is shown to offer a non-perturbing method for following coiled coil formation and for accurately determining dissociation constants. The method is validated using a designed heterodimeric coiled coil. TheKDdeduced by fluorescence monitored titration is in excellent agreement with the value deduced from concentration dependent CD measurements to within the uncertainty of the measurement. However, the fluorescence approach requires less protein, is less time-consuming, can be applied to lower concentrations and could be applied to high throughput screens.