Changes in Secondary Structure Upon Pr to Pfr Transition in Cyanobacterial Phytochrome Cph1 Detected by DNP NMR

Phytochromes perceive subtle changes in the light environment and convert them into biological signals by photoconversion between the red-light absorbing (Pr) and the far-red-absorbing (Pfr) states. In the primitive bacteriophytochromes this includes refolding of a tongue-like hairpin loop close to the chromophore, one strand of an antiparallel β-sheet being replaced by an α-helix. However, the strand sequence in the cyanobacterial phytochrome Cph1 is different from that of previously investigated bacteriophytochromes and has a higher β-sheet propensity. We confirm here the transition experimentally and estimate minimum helix length using dynamic nuclear polarisation (DNP) magic angle spinning NMR. Sample conditions were optimized for protein DNP NMR studies at high field, yielding Boltzmann enhancements ϵ_B of 19 at an NMR field of 18.801 T. Selective labelling of Trp, Ile, Arg, and Val residues with ¹³C and ¹⁵N enabled filtering for pairs of labelled amino acids by the 3D CANCOCA technique to identify signals of the motif ₄₈₃Ile-Val-Arg₄₈₅ (IVR) present in both sheet and helix. Those signals were assigned for the Pfr state of the protein. Based on the chemical shift pattern, we confirm for Cph1 the formation of a helix covering the IVR motif.