Lyophilization Reveals a Multitude of Structural Conformations in the Chromophore of a Cph2-like Phytochrome.

Cyanobacteria sense and respond to various colors of light employing a large number of bilin-based phytochrome-like photoreceptors. All2699 from Nostoc 7120 has three consecutive GAF domains with GAF1 and GAF3 binding a phycocyanobilin chromophore. GAF1, even when expressed independently, can be photoconverted between red-absorbing Pr and far-red-absorbing Pfr states, while the nonphotosensory GAF2 domain is structurally and functionally homologous to the PHY domains in canonical and Cph2-like phytochromes. Here, we characterize possible bilin chromophore conformers using solid-state NMR spectroscopy on the two lyophilized All2699 samples (GAF1-only and GAF1-PHY constructs). On the basis of complete 1H, 13C, and 15N assignments for the chromophore obtained on the two Pr lyophilizates, multiple static conformations of the chromophore in both cases are identified. Moreover, most atoms of the chromophore in the bidomain sample show only subtle changes in the mean chemical shifts relative to those in frozen solution (FS), indicating an optimized interaction of the GAF2 domain with the GAF1-bound chromophore. Our results confirm the conservation of key chromophore-protein interactions and the photoreversibility in both All2699 lyophilizates, offering the possibility to investigate conformational distributions of the heterogeneous chromophore and its functional consequences in phytochromes and other bilin-dependent photoreceptors intractable by the solid-state NMR technique as FSs.