Expanding the chemistry of fluorescent protein biosensors through genetic incorporation of unnatural amino acids.

Fluorescent proteins are essential tools in biological research, ranging from the study of individual biological components to the interrogation of complex cellular systems. Fluorescent protein derived biosensors are increasingly applied to the study of biological molecules and events in living cells. The present review focuses on a specific class of fluorescent protein biosensors in which a genetically installed unnatural amino acid (UAA*) acts as the sensing element. Upon direct interaction with the analyte of interest, the chemical and/or physical properties of UAA* are altered, which triggers fluorescence property changes of the biosensor and generates readouts. In comparison to mutagenesis approaches within the standard genetic code, introduction of UAA*s with a unique functionality and chemical reactivity could broaden the scope of analytes and improve the specificity of biosensors. Nonconventional functional groups in fluorescent proteins enable sensor designs that are not readily accessible using the common twenty amino acids. Recent reports of UAA*-containing fluorescent protein sensors serve as excellent examples for the utility of such sensor design. We envisage that the integration of the two powerful chemical biology tools, fluorescent protein sensors and genetic incorporation of UAA*s, will lead to novel biosensors that can expand and deepen current understanding of cellular processes.