Study of Photostate-Stabilizing Mutants of the Bacteriophytochrome from Xanthomonas campestris

SÁNCHEZ-LAMAS, M.; GOLBAUM, F.A.; RINALDI, J.; ANTELO, G.A.; CONFORTE, V.; OTERO, L.H.; KLINKE, S.; MALAMUD, F.; BONOMI, H.R.

Paraná

Congreso; LIV Reunión Anual de SAIB; 2018

SAIB

Bacteriophytochromes (BphPs) are part of the phytochrome superfamily of photoreceptors. BphPs are proteins that bind biliverdin (BV) as their chromophore and typically present two photostates: a primarily red-absorbing form (Pr) and a far-red-absorbing form (Pfr).We´ve recently shown that the BphP from the plant pathogen bacteria Xanthomonas campestris (XccBphP) modulates the interaction with its host1, and performed several biophysical studies showing that XccBphP is a bathy-like phytochrome (Pfr-enriched equilibrium as ground-state). Moreover, we´ve resolved the 3D crystal structure of the full-length protein in the Pr state (a first in photobiology)2.In order to evaluate the biological role of XccBphP in vivo and resolve its structure in Pfr form, we´ve design punctual and randomized mutants of XccBphP in conserved residues linked to the photochemical behavior. By means of a UV-VIS spectroscopy protocol designed for a rapid and precise characterization of this mutants, we´ve characterized several mutants with different photochemical behavior, including one that stabilizes the Pfr.Several biophysical studies have been made with this mutant, including crystallogenesis and X-ray diffraction. The latter allowed us to obtain the first Pfr structure of a full-length phytochrome to compare it with the already solved Pr. Here, we show results of all these experiments, as well as some preliminary work done in vivo. We provide new insights on the structural mechanisms involved in the light-induced signal transduction of phytochromes.