Taki Masumi, Hohsaka Takahiro, Murakami Hiroshi, Taira Kazunari, Sisido Masahiko
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
J Am Chem Soc. 2002 Dec 11;124(49):14586-90. doi: 10.1021/ja017714+.
Four-base codon strategy was applied to incorporate a fluorophore-quencher pair into specific positions on a single protein; beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap) was employed as a fluorophore and p-nitrophenylalanine (ntrPhe) as a quencher. Their positions were directed by the CGGG/CCCG and GGGC/CCCG four-base codon/anticodon pairs and two doubly mutated streptavidins, i.e., ((52)atnDap, (84)ntrPhe) and ((54)ntrPhe, (84)atnDap) mutants were synthesized through Escherichia coli in vitro protein synthesizing systems. Intramolecular photoinduced electron transfer (ET) was observed as the decrease of intensity in steady-state fluorescence spectroscopy and as the shortening of fluorescence decaytimes. The quenching data indicated that the ET rate reflects the detailed structure of the protein.
四碱基密码子策略被用于将荧光团-猝灭剂对掺入单个蛋白质的特定位置；β-邻氨基苯甲酰-L-α,β-二氨基丙酸（atnDap）用作荧光团，对硝基苯丙氨酸（ntrPhe）用作猝灭剂。它们的位置由CGGG/CCCG和GGGC/CCCG四碱基密码子/反密码子对指导，并且通过大肠杆菌体外蛋白质合成系统合成了两种双突变链霉亲和素，即((52)atnDap, (84)ntrPhe)和((54)ntrPhe, (84)atnDap)突变体。在稳态荧光光谱中观察到分子内光诱导电子转移（ET）表现为强度降低，在荧光衰减时间中表现为缩短。猝灭数据表明ET速率反映了蛋白质的详细结构。
