DOMAIN-SPECIFIC INCORPORATION OF NONINVASIVE OPTICAL PROBES INTO RECOMB PROTEIN

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。描述了一种集成方法，其允许将光学探针域特异性地掺入到大的重组蛋白中。该策略是两种现有技术的组合，表达的蛋白质连接（EPL）和色氨酸（Trp）类似物的体内氨基酸取代的色氨酸。来自c-Crk-I衔接蛋白的Src同源性3（SH 3）结构域已使用大肠杆菌Trp营养缺陷型用Trp类似物7-氮杂色氨酸（7AW）标记。结构、生物化学和热力学研究表明，7AW的掺入不会显著干扰分离结构域的结构或功能。通过EPL将7AW标记的SH 3结构域连接到c-Crk-I Src同源2（SH 2）结构域，产生具有结构域特异性标记的多结构域蛋白c-Crk-I。这种标记的蛋白质的研究表明，SH 3结构域的生化和热力学性质不改变的背景下，一个更大的多结构域蛋白质。这里描述的技术可能是一个有用的工具，在提高我们的理解模块化结构域在其自然环境中的行为，在多结构域蛋白质。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An integrated approach is described that allows the domain-specific incorporation of optical probes into large recombinant proteins. The strategy is the combination of two existing techniques, expressed protein ligation (EPL) and in vivo amino acid replacement of tryptophans with tryptophan (Trp) analogues. The Src homology 3 (SH3) domain from the c-Crk-I adaptor protein has been labeled with a Trp analogue, 7-azatryptophan (7AW), using Escherichia coli Trp auxotrophs. Structural, biochemical, and thermodynamic studies show that incorporation of 7AW does not significantly perturb the structure or function of the isolated domain. Ligation of the 7AW-labeled SH3 domain to the c-Crk-I Src homology 2 (SH2) domain, via EPL, generated the multidomain protein, c-Crk-I, with a domain-specific label. Studies of this labeled protein show that the biochemical and thermodynamic properties of the SH3 domain do not change within the context of a larger multidomain protein. The technology described here is likely to be a useful tool in enhancing our understanding of the behavior of modular domains in their natural context, within multidomain proteins.