Refolding of the Deinococcus Radiodurans phytochrome photosensory module and an extended backbone resonance assignment by solution NMR

Abstract

Solution NMR reveals the structure and dynamics of biomolecules in solution. In particular, the method can detect changes due to perturbation of the molecules, without limiting effects of frozen particles or crystal environments. Phytochromes are photosensors which control the response to red/far-red light in bacteria, fungi and plants, undergo specific structural changes when photoactivated from the Pr to the Pfr state. While structures of phytochromes have been revealed in both states, the structural mechanism of photoconversion remains incompletely understood. Our previous NMR studies of the entire photosensory core module of the D. radiodurans phytochrome have revealed novel structural changes, but the backbone assignment was incomplete. In particular, a lack of the assignment in the protein core hindered more detailed insight in signaling mechanism. Here, we outline an efficient procedure for the refolding of the three-domain, photosensory core fragment of the D. radiodurans phytochrome in its monomeric form. We find that treatment with guanidinium hydrochloride and subsequent dilution effectively refolds the phytochrome, maintaining its functionality. We characterize the refolded protein with solution NMR spectroscopy newly assigning 27 (44) residues in Pr (Pfr), out of which 12 exhibit notable chemical shift perturbation upon photoactivation. The study presents a functional method for purification and refolding of a multidomain protein and opens the door for further structural and dynamic analysis of phytochromes.

Author summary

Refolding of proteins is an established method to increase the deuterium-hydrogen exchange of amid bonds in isotopically labeled proteins, which are located deep in the protein core. Yet, the method has to be optimized for each individual protein and in particular for multidomain proteins it is not trivial to find satisfactory experimental conditions. Here we identify a method to refold a D. radiodurans phytochrome construct and characterize the outcome of the procedure using solution NMR and optical spectroscopy. The quick accessibility on whether the refolded phytochrome was functional or not has been obtained from optical spectra, which also made the screening of a number of additives possible. The procedure led to a significant increase in the number of the assigned residues especially in the protein core, close to the photochemically active chromophore, which enables a more detailed investigation of the structure and dynamics throughout the photocycle of the phytochrome.