Protein Mobility Measurements through Oxidative Green-to-Red Photoconversion of EGFP

Protein dynamics plays a key role in live cell functioning, stimulating the development of new experimental techniques for studying protein transport phenomena. Here, we introduce a relaxation method that is based on the rapid formation of a nonequilibrium concentration profile of the enhanced green fluorescent protein (EGFP) across a sample by its oxidative green-to-red photoconversion. Following the blue-light irradiation of a part of a sample containing EGFP and an oxidant, the diffusion-controlled response of a system is monitored. Changes in the concentration of the initial green-emitting and oxidized red-emitting forms are simultaneously tracked by fluorescence lifetime measurements using the time-correlated single photon counting. We show that the diffusion coefficient of EGFP in water, determined by this method, is in good agreement with previously published data. This approach opens a way for the studies of intracellular viscosity changes combined with sensing of elevated levels of reactive oxygen species.