Position‐Specific Incorporation of Fluorescent Non‐natural Amino Acids into Maltose‐Binding Protein for Detection of Ligand Binding by FRET and Fluorescence Quenching

FRETting about MBP: Position‐specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. Here we demonstrate that position‐specific incorporation of fluorescent non‐natural amino acids in response to expanded codons enables us to detect ligand‐binding activity of maltose‐binding protein (MBP) through fluorescence resonance energy transfer (FRET) and ligand‐dependent fluorescence quenching. magnified image 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.