Expression, purification and crystallization of the photosensory module of phytochrome B (phyB) from Sorghum bicolor

Sorghum, a short-day tropical plant, has been adapted for temperate grain production, in particular through the selection of variants at the MATURITY loci (Ma1-Ma6) that reduce photoperiod sensitivity. Ma3 encodes phytochrome B (phyB), a red/far-red photochromic biliprotein photoreceptor. The multi-domain gene product, comprising 1178 amino acids, autocatalytically binds the phytochromobilin chromophore to form the photoactive holophytochrome (Sb.phyB). This study describes the development of an efficient heterologous overproduction system which allows the production of large quantities of various holoprotein constructs, along with purification and crystallization procedures. Crystals of the Pr (red-light-absorbing) forms of NPGP, PGP and PG (residues 1-655, 114-655 and 114-458, respectively), each C-terminally tagged with His₆, were successfully produced. While NPGP crystals did not diffract, those of PGP and PG diffracted to 6 and 2.1 Å resolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8 Å resolution. These results demonstrate that the diffraction quality of challenging protein crystals can be improved by removing flexible regions, shifting fusion tags and altering small-molecule ligands.