Premium
A Spectroscopy‐based Methodology for Rapid Screening and Characterization of Phytochrome Photochemistry in Search of Pfr‐favored Variants
Author(s) -
Antelo Giuliano Tomás,
SánchezLamas Maximiliano,
Goldbaum Fernando Alberto,
Otero Lisandro Horacio,
Bonomi Hernán Ruy,
Rinaldi Jimena
Publication year - 2020
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/php.13313
Subject(s) - phytochrome , chromophore , biliverdin , xanthomonas campestris , spectroscopy , chemistry , characterization (materials science) , photochemistry , botany , materials science , biology , biochemistry , physics , nanotechnology , red light , heme , enzyme , gene , heme oxygenase , quantum mechanics
Phytochromes are photosensitive proteins with a covalently bound open‐chain chromophore that can switch between two principal states: red light absorbing Pr and far‐red light absorbing Pfr. Our group has previously shown that the bacteriophytochrome from Xanthomonas campestris pv. campestris ( Xcc BphP) is a bathy‐like phytochrome that uses biliverdin IXα as a co‐factor and is involved in bacterial virulence. To date, the Xcc BphP crystal structure could only be solved in the Pr state, while the structure of its Pfr state remains elusive. The aims of this work were to develop an efficient screening methodology for the rapid characterization and to identify Xcc BphP variants that favor the Pfr form. The screening approach developed here consists in analyzing the UV‐Vis absorption behavior of clarified crude extracts containing recombinant phytochromes. This strategy has allowed us to quickly explore over a hundred Xcc BphP variants, characterize multiple variants and identify Pfr‐favored candidates. The high‐quality data obtained enabled not only a qualitative, but also a quantitative characterization of their photochemistry. This method could be easily adapted to other phytochromes or other photoreceptor families.