Development of specific l-methionine sensors by FRET-based protein engineering.

Abstract

Amino acids are essential nutrients that are not only used as protein building blocks but are also involved in various biochemical processes and in the development of human diseases. Quantitative analysis of amino acids in complex biological samples is an important analytical process used for understanding amino acid biochemistry and diagnosis of human diseases. In this study, a protein sensor based on fluorescence resonance energy transfer (FRET) was designed for the quantitative analysis of l-Met, in which a fluorescent unnatural amino acid (CouA) and YFP were used as a FRET pair. A natural Met-binding protein (MetQ) was chosen as a sensor protein, and CouA and YFP were incorporated into the protein by genetic code expansion technology and genetic fusion. Among the four sites screened for CouA incorporation into MetQ, R189 was selected as the best site for l-Met sensing. The sensor protein (YFP-MetQ-R189CouA) showed a large FRET signal change (2.7-fold increase) upon l-Met binding. To improve amino acid specificity of the sensor protein, the ligand-binding site was engineered, and the mutant sensor (YFP-MetQ-R189CouA-H88F) with the H88F mutation was identified, which showed no FRET signal change with d-Met and l-Gln at 50 μM concentration and retained the maximum FRET signal change with l-Met. The optimized sensor protein was evaluated for biochemical applications. l-Met concentration in FBS and optical purity in a mixture of d- and l-Met were successfully determined. Because l-Met is biochemically important owing to its involvement in cancer cell growth and autophagy, the sensor protein would be useful for quantitative analysis of l-Met in a complex biological sample. In addition, the design strategy used in this study can be applied to other small molecule-binding proteins for the development of protein sensors for important biomolecules.