The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and time-resolved fluorescence spectroscopy regarding its applicability for in vivo measurements. Based on the purified sensor protein, it is shown that the NADH dependence of Frex fluorescence can be described by a Hill function with a concentration of half-maximal sensor response of K D ≈ 4 µM and a Hill coefficient of n ≈ 2. Increasing concentrations of NADH have moderate effects on the fluorescence lifetime of Frex, which changes by a factor of two from about 500 ps in the absence of NADH to 1 ns under fluorescence-saturating NADH concentrations. Therefore, the observed sevenfold rise of the fluorescence intensity is primarily ascribed to amplitude changes. Notably, the dynamic range of Frex sensitivity towards NADH highly depends on the NAD+ concentration, while the apparent K D for NADH is only slightly affected. We found that NAD+ has a strong inhibitory effect on the fluorescence response of Frex during NADH sensing, with an apparent NAD+ dissociation constant of K I ≈ 400 µM. This finding was supported by fluorescence lifetime measurements, which showed that the addition of NAD+ hardly affects the fluorescence lifetime, but rather reduces the number of Frex molecules that are able to bind NADH. Furthermore, the fluorescence responses of Frex to NADH and NAD+ depend critically on pH and temperature. Thus, for in vivo applications of Frex, temperature and pH need to be strictly controlled or considered during data acquisition and analysis. If all these constraints are properly met, Frex fluorescence intensity measurements can be employed to estimate the minimum NADH concentration present within the cell at sufficiently low NAD+ concentrations below 100 µM.
Keywords: Decay-associated spectra; Fluorescence lifetime; Fluorescence sensor protein; Frex; Light-driven biohydrogen production; NAD+; NADH; Redox sensing.