Transient de-protonation of the chromophore affects protein dynamics proximal and distal to the linear tetrapyrrole chromophore in phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes including Cph1 from the cyanobacterium Synechocystis 6803 act as photochemical switches that interconvert between stable red (Pr) and metastable far-red (Pfr) - absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr-state, involving pH-titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain) and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.