Study of photostate-stabilizing mutants of the bacteriophytochrome from Xanthomonas campestris

KLINKE, SEBASTIÁN; MALAMUD, FLORENCIA; BONOMI, HERNAN R.; ANTELO, GIULIANO; CONFORTE, VALERIA; OTERO, LISANDRO HORACIO; SANCHEZ, MAXIMILIANO; GOLDBAUM, FERNANDO A.; RINALDI, JIMENA

Parana

Congreso; 54th Annual Meeting Agentine Society for Biochemistry and Molecular Biology LIV Reunion Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Bacteriophytochromes (BphPs) arepart of the phytochrome superfamily of photoreceptors. BphPs are proteins that bind biliverd in (BV) as their chromophore and  typically  present two  photostates:  a primarily  red-absorbing  form (Pr)  and  a  far-red-absorbing  form (Pfr).We have  recently shown  that the  BphP  from  the  plant pathogen  bacteria Xanthomonas campestris (XccBphP)  modulates  the interaction  with  its host, and performed several biophysical studies showing that XccBphP isa bathy-like phytochrome (Pfr-enriched equilibrium as ground-state). Moreover,we have resolved the 3D crystal structure of the full-length protein in the Pr state (the first in photobiology). In order to evaluate the biological role  of XccBphP in  vivo and  resolve its  structure  in  Pfr form,  we have designed punctual  and randomized  mutants  of XccBphP  in  conserved residues linked to the photochemical behavior. By means of a UV-VIS spectroscopy protocol designed fora rapid and precise characterization of these mutants,  we have 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.<!-- /* Font Definitions */ @font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:1;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:0 0 0 0 0 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:swiss;mso-font-pitch:variable;mso-font-signature:-536859905 -1073732485 9 0 511 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin-top:0cm;margin-right:0cm;margin-bottom:8.0pt;margin-left:0cm;line-height:107%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-fareast-language:EN-US;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-family:"Calibri",sans-serif;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-fareast-language:EN-US;}.MsoPapDefault{mso-style-type:export-only;margin-bottom:8.0pt;line-height:107%;}@page WordSection1{size:595.3pt 841.9pt;margin:70.85pt 3.0cm 70.85pt 3.0cm;mso-header-margin:35.4pt;mso-footer-margin:35.4pt;mso-paper-source:0;}div.WordSection1{page:WordSection1;}-->