Fig. 7: Model of VCF signal generation during CiHv1 conformational changes.
From: Multiple mechanisms contribute to fluorometry signals from the voltage-gated proton channel
The model assumes two major VSD movements among three states traced by the attached TAMRA (orange star). The resting (1), the intermediate (2) and the activated (3) states are associated with different fluorescence intensities (F1, F2, F3) in each construct for a single TAMRA molecule. In the H188A/H179A mutant (top row panels) there is no quenching residue near TAMRA. In response to a depolarizing pulse, the dye first moves away from the lipid bilayer from state 1 to 2 and the fluorescence intensity decreases, since F1 > F2. Subsequently, there is a second motion from state 2 to 3, when the dye approaches the lipid bilayer, and the fluorescence increases to a level above the initial one: F3 > F1 > F2. In single alanine mutants and the CiHv1-E241C there is/are weak quenching residue(s) near the dye (middle row panels). The motion is similar to that in the top row panels, but during the second motion as TAMRA approaches both the quencher and the bilayer, the latter effect overrides the weak effect of the quencher, thus, the fluorescence increases but remains below the original one: F1 > F3 > F2. In the tryptophan mutants (bottom row panels) there is/are strong quenching residue(s) near the dye. During depolarization, it moves similarly to the top row panel motion, away from the membrane and toward the quenching residue from state 1 to 2 and the fluorescence intensity decreases, F1 > F2. Subsequently, in the second motion from state 2 to 3, TAMRA approaches both the membrane and the quencher, the latter effect overrides the lipid effect and fluorescence further decreases: F1 > F2 > F3.
