Position-specific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anticodon pairs.
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.
Taki
Masumi
M
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Hohsaka
Takahiro
T
Murakami
Hiroshi
H
Taira
Kazunari
K
Sisido
Masahiko
M
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000926:Anticodon / Q000235:genetics*; D001710:Biotin / Q000737:chemistry; D003062:Codon / Q000235:genetics*; D003198:Computer Simulation; D004583:Electrons; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D013050:Spectrometry, Fluorescence; D019809:Streptavidin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D062367:ortho-Aminobenzoates / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D000926:Anticodon; D003062:Codon; D005456:Fluorescent Dyes; C439761:beta-anthraniloyl-amino-beta-diaminopropanoic acid; D062367:ortho-Aminobenzoates; C040239:4-nitrophenylalanine; D010649:Phenylalanine; D001710:Biotin; D019809:Streptavidin