Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure
Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure
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色氨酸荧光的硒代蛋氨酸淬灭提供了蛋白质结构的简单探针
DOI：
10.1021/acs.biochem.6b01000
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发表时间：
2017
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;Zou, Junjie;Bilsel, Osman;Raleigh, Daniel P.
荧光光谱法是研究蛋白质折叠、蛋白质-配体相互作用和蛋白质动力学的最广泛使用的方法之一。色氨酸通常是选择的荧光团，因为其对环境的敏感性和具有天然氨基酸的最高量子产率;然而，色氨酸荧光的变化可能难以解释特定的结构变化。色氨酸荧光猝灭剂的引入可以提供关于特定结构的信息，特别是如果猝灭是短程的;然而，最常用的猝灭剂是组氨酸，并且它仅在咪唑侧链质子化时有效，因此限制了可以采用该方法的pH范围。此外，组氨酸并不总是保守取代，如果插入蛋白质的疏水核心，可能会不稳定。在这里，我们说明了使用的色氨酸-硒代蛋氨酸（MSe）对作为蛋白质结构的特异性探针。MSe需要与Trp紧密接触以淬灭其荧光，并且可以利用这种效应来设计α-螺旋和β-折叠形成的特异性探针。该方法说明使用平衡和时间分辨荧光测量的设计肽和球状蛋白质。MSe很容易被整合到蛋白质中，并为疏水侧链提供保守的替代，并且Trp荧光的MSequenching不依赖于pH。MSe的氧化形式，硒代甲硫氨酸硒氧化物，也是Trp荧光的有效淬灭剂。
Fluorescence spectroscopy, relying on intrinsic protein fluorophores, is one of the most widely used methods for studying protein folding, protein–ligand interactions, and protein dynamics. Tryptophan is usually the fluorophore of choice, given its sensitivity to its environment and having the highest quantum yield of the natural amino acids; however, changes in tryptophan fluorescence can be difficult to interpret in terms of specific structural changes. The introduction of quenchers of tryptophan fluorescence can provide information about specific structures, particularly if quenching is short-range; however, the most commonly employed quencher is histidine, and it is effective only when the imidazole side chain is protonated, thus limiting the pH range over which this approach can be employed. In addition, histidine is not always a conservative substitution and is likely to be destabilizing if inserted into the hydrophobic core of proteins. Here we illustrate the use of a Trp-selenomethionine (MSe) pair as a specific probe of protein structure. MSerequires a close approach to Trp to quench its fluorescence, and this effect can be exploited to design specific probes of α-helix and β-sheet formation. The approach is illustrated using equilibrium and time-resolved fluorescence measurements of designed peptides and globular proteins. MSeis easily incorporated into proteins and provides a conservative replacement for hydrophobic side chains, and MSequenching of Trp fluorescence is pH-independent. The oxidized form of MSe, selenomethionine selenoxide, is also an efficient quencher of Trp fluorescence.