Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.
J Am Chem Soc. 2012 Nov 21;134(46):18883-5. doi: 10.1021/ja307179q. Epub 2012 Nov 8.
Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl-tRNA(CUA). Excimer formation (λ(ex) 342 nm; λ(em) 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.
由于现有 FRET 对距离的微小变化缺乏敏感性（这是由酶的尺寸决定的限制），因此制备了含有两个芘基部分的 DHFR，以能够观察到激基缔合物的形成。使用体外表达系统，在存在芘-1-丙氨酰-tRNA（CUA）的情况下，将芘-1-丙氨酸引入 DHFR 的 16 位和 49 位。在保留其催化能力的修饰后的 DHFR 中观察到激基缔合物的形成（λ（ex）342nm；λ（em）481nm），并在多次和单次周转条件下进行了研究。激基缔合物的形成似乎是在 H 转移后伴随着蛋白质构象的变化，涉及芘基部分的相对位置和取向，并且可能与产物的解离有关。
