Incorporation of unnatural amino acid and its application to effective single-molecule analysis

Abstract

Beyond natural 20 amino acids, unnatural amino acids having novel functional group have been consolidated into the existing genetic code as a 21st genetic code. Applications of genetically encoded unnatural amino acids enable new means for tailored manipulation of proteins, such as site-specific labeling or modification of protein, probing protein structure and function, identifying and regulating protein activity, and generating proteins with new properties. In this study, we used genetic code expansion technique for effective surface-based protein analysis, especially single-molecule analysis. Immobilization of proteins in a functionally active form and proper orientation is crucial for effective surface-based analysis of proteins. Here we present a general method for controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid and click chemistry. The utility and potential of this method was demonstrated by applying it to the analysis of interaction between a pathogenic protein DrrA of Legionella pneumophila and its binding partner Rab1 of human. Kinetic analysis using Surface Plasmon Resonance revealed that immobilization of site-specifically biotinylated DrrA results in about 10-fold higher sensitivity in binding assay than the conventional immobilization of DrrA with random orientation. The present method is expected to find wide applications in the fields of the surface-based studies of protein-protein (or ligand) interactions, drug screening, biochip, and single molecule analysis. For effective single-molecule Fluorescence Resonance Energy Transfer (smFRET) analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. Here we showed a case that site-specific labeling of protein via incorporation of unnatural amino acid gives rise to high distributional homogeneity in the FRET efficiency histogram compared to the conventional method using double cysteines...