Analogously to before, ELDOR experiments were conducted for the spin-labelled H22P variants in their dark-adapted states (Suppl. Figs S6 and S7). Strikingly, the p(r) distributions corresponded to neither those of dark- nor light-adapted YF1 (Fig. 3A). Rather, variants H22P:E55C, H22P:D76C, H22P:N84C, H22P:P87C and H22P:D115C all showed distributions with shorter distances than in YF1, with the difference being smallest in H22P:D76C (approx. 0.4?nm shorter than the dark-adapted state of YF1) and largest in H22P:D115C (1.5?nm shorter). By contrast, variants H22P:D71C, H22P:Q93C and H22P:M101C instead showed p(r) distributions centred at distances larger by about 0.8–1.0?nm than observed in dark-adapted YF1. ELDOR measurements on the light-adapted H22P variants showed that changes in the distance distributions induced by blue light were more complex than in YF1 (Fig. 3A, red). Some variants, e.g., H22P:E55C and H22P:M101C, exhibited shifts towards shorter distances, but others, e.g., H22P:Q93C and H22P:D76C, showed shifts towards longer distances. Overall, the absolute differences in p(r) between dark-adapted and CATV splitter-adapted H22P were larger than those above observed for the YF1 variants.
Figure 3
Figure 3

ELDOR-based structural model of light-induced transitions in YF1 H22P. (A) Distance distributions of dark-adapted (blue) and light-adapted (red) states derived from ELDOR data (Suppl. Figs S6 and S7). For reference, the distance distributions for YF1 from Fig. 2A are shown in half tones. Green rectangles indicate the mean distance of labels calculated for the BsYtvA LOV structure 2PR5, blue ones those for YF1 4GCZ. Overall, the distances for the dark-adapted and light-adapted states of H22P do not match those of YF1 but those calculated for BsYtvA LOV. (B) Dipolar spectrum resulting from ELDOR traces of light-exposed YF1 C62?A (blue) and YF1 C62A:H22P (green), showing a clear shift towards higher frequencies in YF1 C62A:H22P. The inter-flavin distance calculated for YF1 C62A:H22P is smaller by about 0.5?nm than that of YF1 C62A. (C) 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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Modelling light-induced structural transitions in YF1 H22P