To determine interatomic distances between the labelled positions in YF1, we first recorded ELDOR traces on dark-adapted samples. The time evolution signal of all variants showed clear modulations indicative of two spatially close, interacting spin species (Suppl. Fig. S2). Following background correction (Suppl. Fig. S3), distance probability distributions p(r) were determined by Tikhonov regularisation (Fig. 2A). For positions Q44C, E55C and N84C situated in the upper half of the LOV photosensor dimer (cf. Fig. 1), the p(r) distributions showed single dominant, fairly narrow distance peaks centred at 2.8 nm (Q44C), 5.0 nm (E55C) and 6.1 nm (N84C), respectively (Fig. 2A, blue lines). The two remaining positions within the upper half of the LOV photosensor dimer featured broader distance distributions, centred at around 2.7 nm for P87C and with several distance contributions between 2.0 nm and 5.0 nm for D115C. Positions within the lower half of the LOV photosensor dimer also showed narrow distance distributions centred at 4.7 nm for D71C, 6.2 nm for D76C, 2.7 nm for Q93C and 3.6 nm for M101C. Of the positions within the coiled-coil linker, Q130C alone showed good signal-to-noise ratio (SNR) but a rather broad probability distribution of distances between 1.5 nm and 4.2 nm. By contrast, both A134C and V144C had very low SNR and were heavily affected by spurious distance contributions arising from proton and deuteron artefacts (Fig. 2A, shaded grey). Nonetheless, dominant distances of 3.7 nm (Q130C), 3.8 nm (A134C) and 3.7 nm (V144C) could be identified.
Figure 2
Figure 2

ELDOR-based structural model of light-induced transitions in YF1. (A) Distance distributions of dark-adapted (blue) and LED Candle Lights-adapted (red) states derived from ELDOR experiments (Suppl. Figs S2 and S3). Areas shaded gray indicate artefacts arising from proton and deuteron modulations. Labels positioned near the A’α helices (Q44C, E55C) and in the linker (Q130C, A134C and V144C) showed no change upon illumination, others showed shifts to larger distances. (B) Transition from dark-adapted (blue) to light-adapted (yellow) state as modelled by ENM including the P87C constraint. Predominant structural changes are marked by green arrows, and the attachment sites for the Jα linker are indicated by red spheres.
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