Determination of Protein Complexes with NADH in Live Cells

Abstract

The ratio of free/bound NADH is used to measure the redox state of the cells. This ratio does not provide specific information about which proteins are involved in binding of NADH. In vitro, FRET between tryptophan containing proteins (excited between 280-300 nm) and the NADH co-factor (emitting between 400-450 nm) has been exploited to detect the ratio of free/bound NADH to specific proteins. In live cell microscopy this approach is difficult to implement because it requires UV excitation. If a specific protein interacting with NADH has an absorption or fluorescent moiety, this will result in the quenching of the NADH fluorescence. However, since there is a large amount of NADH, this decrease of fluorescence is difficult to associate to the specific protein. Instead, when FRET occurs between NADH and a fluorescently tagged protein there will be a sensitized emission of the acceptor molecule which is easy to detect. Here we develop an in vivo FLIM/FRET assay in which we use FRET between the NADH (using 2-photon excitation at 740 nm) and the green fluorescent protein attached to an NADH binding protein. In our assay, we use the histone 2B-EGFP construct which is localized in the nucleus and has been shown that this tagged protein does not modify its activity. This FLIM/FRET assay can be used to determine transcription factor binding partners of NADH if the transcription factor is labeled with a fluorescent protein. In addition the FLIM data unequivocally identifies the emission of the GFP from other intrinsic autofluorescent signals (FAD). The phasor lifetime map of histone bound NADH is generated to show areas of increased chromatin activity. NADH is excited in the UV using 2-photon excitation. Thi work is supported in part by NIH-P41 P41-RRO3155, 8P41GM103540 and P50-GM076516.