Real-time mapping of protein proton dynamics through photoresponsive non-canonical amino acids and ultrafast spectroscopy

Proton transfer (PT) underpins a wide range of protein functions in biology, including bioenergetics, enzymatic catalysis, pH regulation, protein stability, membrane transport, and signal transduction. Despite its central role, directly mapping PT pathways inside proteins remains a major challenge, as current methods typically capture only the end products of PT rather than the transfer process itself. TrackingProtons seeks to overcome this limitation by establishing a new strategy to initiate and monitor PT in real time using genetically encoded, light-sensitive non-canonical amino acids (ncAAs). These ncAAs act as photoacids and photobases that undergo large, reversible pKa shifts upon optical excitation, creating a strong driving force for PT between defined sites within proteins. The project will develop an orthogonal genetic code expansion system to enable site-specific dual incorporation of a proton donor and acceptor into the same protein scaffold. Carbonic anhydrase, a prototypical PT enzyme, will serve as the model system, with PT dynamics tracked using complementary ultrafast fluorescence and infrared spectroscopy. This approach will provide the first direct observation of PT trajectories with molecular precision, offering transformative insights into proton-coupled processes across biology. Beyond advancing fundamental understanding, the project will establish expertise at the interface of protein engineering and ultrafast spectroscopy, supporting the applicant’s progression toward scientific independence.