Distance mapping in proteins using fluorescence spectroscopy: the tryptophan-induced quenching (TrIQ) method.
Distance mapping in proteins using fluorescence spectroscopy: the tryptophan-induced quenching (TrIQ) method.
复制标题
DOI：
10.1021/bi100907m
复制
发表时间：
2010-11-16
期刊：
影响因子：
2.9
通讯作者：
Farrens, David L.
中科院分区：
文献类型：
--
作者：
Mansoor, Steven E.;DeWitt, Mark A.;Farrens, David L.
研究蛋白质结构和功能之间的相互作用仍然是一项艰巨的任务。特别缺乏的是真实的时间测量结构变化的方法。在这里，我们报告我们最近的改进，可以用来解决这些问题的方法。这种方法，我们现在称之为色氨酸诱导淬火（TrIQ），提供了一个简单，灵敏和廉价的方法来解决构象动力学和短程蛋白质相互作用的问题。重要的是，TrIQ只发生在相对较短的距离（约5至15 μ m），使其成为传统荧光共振能量转移（FRET）方法的补充，这些方法发生的距离太大，无法精确研究蛋白质结构。顾名思义，TrIQ测量色氨酸（Trp）在某些荧光团中诱导的有效淬灭。我们在这里提出了我们的分析的TrIQ效应的五种不同的荧光团，跨越一系列的大小和光谱特性。每个探针连接到T4溶菌酶上的四个不同的半胱氨酸残基，并测量由附近的Trp引起的TrIQ的程度。我们的研究结果表明，对于较小的探头，TrIQ是距离依赖的。此外，我们还演示了如何TrIQ数据可以进行分析，以确定参与静态，非荧光复合物与色氨酸的荧光团的分数。基于这种分析，我们的研究表明，每个荧光团具有不同的TrIQ配置文件，或“球淬灭”，这与它的大小，旋转灵活性，和连接接头的长度。这种基于TrIQ的“淬灭球”对每个Trp探针对都是独特的，并反映了人们可以预期看到TrIQ效应的距离。它提供了一个简单的，容易获得的方法，映射蛋白质内的距离和监测构象变化，使用荧光光谱。
Studying the interplay between protein structure and function remains a daunting task. Especially lacking are methods for measuring structural changes in real time. Here we report our most recent improvements to a method that can be used to address such questions. This method, which we now call Tryptophan induced quenching (TrIQ), provides a straightforward, sensitive and inexpensive way to address questions of conformational dynamics and short-range protein interactions. Importantly, TrIQ only occurs over relatively short distances (~5 to 15 Å), making it complementary to traditional fluorescence resonance energy transfer (FRET) methods that occur over distances too large for precise studies of protein structure. As implied in the name, TrIQ measures the efficient quenching induced in some fluorophores by tryptophan (Trp). We present here our analysis of the TrIQ effect for five different fluorophores that span a range of sizes and spectral properties. Each probe was attached to four different cysteine residues on T4 lysozyme and the extent of TrIQ caused by a nearby Trp was measured. Our results show that for smaller probes, TrIQ is distance dependent. Moreover, we also demonstrate how TrIQ data can be analyzed to determine the fraction of fluorophores involved in a static, non-fluorescent complex with Trp. Based on this analysis, our study shows that each fluorophore has a different TrIQ profile, or "sphere of quenching", which correlates with its size, rotational flexibility, and the length of attachment linker. This TrIQ-based "sphere of quenching" is unique to every Trp-probe pair and reflects the distance within which one can expect to see the TrIQ effect. It provides a straightforward, readily accessible approach for mapping distances within proteins and monitoring conformational changes using fluorescence spectroscopy.
登录
查看更多内容
影响因子：
15
作者：
Borbat, PP;Mchaourab, HS;Freed, JH
通讯作者：
Freed, JH
影响因子：
5.6
作者：
Klare, JP;Bordignon, E;Engelhard, M
通讯作者：
Engelhard, M
影响因子：
4.8
作者：
Janz, JM;Farrens, DL
通讯作者：
Farrens, DL
影响因子：
4.7
作者：
Marmé, N;Knemeyer, JP;Wolfrum, J
通讯作者：
Wolfrum, J
影响因子：
2.9
作者：
Mansoor, SE;Mchaourab, HS;Farrens, DL
通讯作者：
Farrens, DL