Selective substitution of tryptophan analogs in recombinant proteins for fluorescence studies of protein-protein interactions

一些生物和物种的基因组现已被揭示。这些基因组的基因产物的一级序列，大多数是蛋白质，许多未知的结构和功能，已被确定。人们对蛋白质之间或与DNA或RNA之间的相互作用和复合物如何导致生理功能或事件非常感兴趣。蛋白质荧光性质的信息含量不仅可以揭示蛋白质复合物相互作用片段的重要结构和动力学信息，还可以揭示非相互作用片段的构象结构。这些荧光研究通常利用芳香族氨基酸色氨酸（Trp）的固有荧光。然而，当蛋白质复合物中存在许多Trp残基时，不可能解开荧光性质并将变化归因于单个Trp残基。本研究提案描述了如何使用体外方法和无细胞蛋白质合成，用光谱学上不同的色氨酸类似物（如5-羟基色氨酸）取代单个色氨酸残基。

The genomes of several organisms and species have now been revealed. The primary sequences of the gene products of these genomes, the majority being proteins, many of unknown structure and function, have been identified. There is considerable interest in how the interactions and complexes of proteins with one another or with DNA or RNA result in physiological functionality or events. It has been shown that the information content of the fluorescence properties of proteins can reveal important structural and dynamic information of not only the interacting segments of protein complexes by also the conformational structures of the non-interacting segments. These fluorescence studies often take advantage of the intrinsic fluorescence of the aromatic amino acid, tryptophan (Trp). However, when there are a number of Trp residues in the protein complex it is not possible to unravel the fluorescence properties and attribute changes to a single Trp residue. This research proposal describes how a single Trp residue may be substituted by a spectroscopically distinct tryptophan analogue such as 5-hydroxyTrp using in vitro methods and cell free protein synthesis.