Acidic pH, Ca²⁺ and target membrane control GP conformational dynamics.

(A) Experimental setup for smFRET imaging of pseudovirions labelled with Cy3 and Cy5 fluorophores. Labelled pseudovirions were immobilized on a passivated quartz microscope slide via a biotin-streptavidin linkage and imaged using prism-based TIRF microscopy. (B) Fluorophore attachment positions (red and green circles; residues 501 and 610 in GP2) are indicated on a GP protomer (PDB: 5JQ3). The indicated distance of 30 Å was previously determined through MD simulation of the fluorophore-labelled trimer [12]. (C) Representative FRET trajectories (blue) of GP^CL overlaid with idealization (red) determined through HMM analysis. The trajectory acquired at pH 7.5 (top) shows transitions between the pre-fusion conformation (0.92 ± 0.08 FRET), two intermediate-FRET states (0.72 ± 0.08 and 0.49 ± 0.08), and a low-FRET state (0.24 ± 0.08). In contrast, the FRET trajectory acquired in the presence of liposomes at pH 5.5 and 1 mM Ca²⁺ shows predominantly low FRET indicating a conformation stabilized by the presence of a target membrane. (D) Histograms of experimentally observed FRET values for wild-type GP^CL under the conditions indicated. Error bars indicate the mean probability per bin determined from three independent groups of traces. Overlaid on the histograms are single Gaussian distributions (grey, shaded) indicating the FRET states observed, which were generated during HMM analysis of the individual FRET trajectories. The sum of the four Gaussian distributions is also overlaid in red. The zero-FRET state, which arises due to photobleaching of the fluorophores is omitted to enable accurate visualization of the low-FRET state occupancy. (E) Violin plots indicating the distribution of occupancy in the high-FRET pre-fusion (top) and low-FRET (bottom) states. Overlaid on each plot is the median (circle), mean (horizontal line), and 25^th and 75^th quantile (vertical lines). Indicated p-values were determined by one-way ANOVA (ns, not significant). (F) TDPs displaying the relative frequency of transitions of GP^CL under the conditions indicated. Specific transitions between the observed FRET states are labelled with dashed circles.