Abstract
Dopamine (DA) system impairment in the central nervous system links to both motor and mental dysfunction in human. However, it remains impossible to monitor the brain DA dynamic in cellular level. MAO-B (monoamine oxidase B) is an enzyme anchored on the mitochondrial outer membrane to catalyse amine oxidation, including DA; a process requires FAD as a cofactor. Because of MAO-B absorbance at 400-500 nm spectrums, MAO-B fused with a GFP chromophore cannot be elicited by 488 nm light if FAD remains in oxidized form, we called this phenomenon as “Shield effect”. Shield effect would be diminished upon the enzyme binds to the substrate, which would allow us to “visualize” the substrate binding of MAO-B in real time. Indeed, we have established such a molecular probe, MMG1, which could detect the intracellular fluctuation of DA concentration by monitoring GFP fluorescent readout. The L-DOPA (dopamine precursor)–induced fluorescence in a set of quantitative experiments using flow cytometry confirmed that MMG1 is sensitive to L-DOPA in a dose-dependent manner. To ultimately validate this DA probe, I constructed three MMG1 mutants, MMG1Y398S, MMG1Y435S, and MMG1C397A, which are defective in FAD or substrate binding. To further characterize this DA probe (MMG1), I characterized the enzyme kinetics of MMG1 and revealed the substrate binding affinity of MMG1 is comparable to native MAO-B. Together, this study provides the first in vivo tool that could monitor DA dynamics in cellular level.