title: Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching.
abstract: Position-specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. We have developed a method for the incorporation of visible-wavelength-fluorescent non-natural amino acids into proteins in a cell-free translation system. Using this technique, we introduced one or two BODIPY-linked amino acids into maltose-binding protein (MBP) to obtain MBP derivatives showing ligand-dependent changes in fluorescence intensity or intensity ratio. BODIPY-FL-aminophenylalanine was incorporated in place of 15 tyrosines, as well as the N-terminal Lys1, and the C-terminal Lys370 of MBP. Fluorescence measurements revealed that MBP containing a BODIPY-FL moiety in place of Tyr210 showed a 13-fold increase in fluorescence upon binding of maltose. Tryptophan-to-phenylalanine substitutions suggest that the increase in fluorescence was the result of a decrease in the quenching of BODIPY-FL by tryptophan located around the binding site. MBP containing a BODIPY-558 moiety also showed a maltose-dependent increase in fluorescence. BODIPY-FL was then additionally incorporated in place of Lys1 of the BODIPY-558-containing MBP as a response to the amber codon. Fluorescence measurements with excitation of BODIPY-FL showed a large change in fluorescence intensity ratio (0.13 to 1.25) upon binding of maltose; this change can be attributed to fluorescence resonance energy transfer (FRET) and maltose-dependent quenching of BODIPY-558. These results demonstrate the usefulness of the position-specific incorporation of fluorescent amino acids in the fluorescence-based detection of protein functions.
authors.affiliation: Japan Advanced Institute of Science and Technology, Asahidai, Nomi, Ishikawa, Japan.
authors.forename: Issei
authors.initials: I
authors.lastname: Iijima
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hohsaka
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001896:Boron Compounds / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D017186:Titrimetry
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000596:Amino Acids; D001896:Boron Compounds; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D058619:Maltose-Binding Proteins; D008320:Maltose