Fluorescent proteins (FPs) have been extensively used in biological research for the study of gene expression, protein function and trafficking, and protein-protein interactions. They have also been engineered to act as biosensing agents to detect intracellular signaling molecules and other small molecule metabolites often associated with the progression of diseases. Although FPs have been engineered extensively to obtain novel functionalities, they are traditionally modified using the 20 canonical amino acids. Thus, limiting the number of functional groups available to design and construct proteins. The expansion of the genetic code through the incorporation of non-canonical amino acids (ncAAs) allows us to introduce amino acids containing novel functional groups to develop FPs with improved or new chemical and photophysical properties. We aim to develop reaction-based biosensors by modifying key residues in the photoconvertable FP, mEos2. Our approach entails the introduction of ncAAs that contain chemical functional groups that modify the ability of FPs to fluoresce and have high reactivity with the analyte of interest. Upon interaction with the analyte, the light-emitting capabilities of the FP are restored or altered. Herein we report the development of a FP-based sensors through the incorporation of p-boronophenylalanine in the chromophore-forming Tyr66 position for the detection of hydrogen peroxide (H2O2). Under the presence of micromolar concentrations of H2O2, our “turn-on” sensor undergoes a dark-to-green change, followed by red photoconversion once irradiated with 405 nm wavelength. This design represents the generation of the first reaction-based photocontrollable FP for hydrogen peroxide detection and future super-resolution imaging.

Engineering fluorescent proteins through non-canonical amino acid incorporation for biosensing and imaging of reactive species

ACS Spring Meeting 2021

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