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The Pseudomonas syringae pv. actinidiae chemoreceptor protein F (PscF) periplasmic sensor domain: cloning, purification and X‐ray crystallographic analysis
Author(s) -
Oulavallickal Tifany,
Brewster Jodi L.,
McKellar James L. O.,
Fairhurst Michael J.,
Tenci Nicholas A.,
Gerth Monica L.
Publication year - 2017
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x17016831
Subject(s) - periplasmic space , lac operon , escherichia coli , pseudomonas syringae , molecular replacement , biology , biochemistry , chemistry , protein structure , gene
Nitrate‐ and nitrite‐sensing (NIT) domains are found associated with a wide variety of bacterial receptors, including chemoreceptors. However, the structure of a chemoreceptor‐associated NIT domain has not yet been characterized. Recently, a chemoreceptor named PscF was identified from the plant pathogen Pseudomonas syringae pv. actinidiae that is predicted to contain a periplasmic NIT domain. The PscF sensor domain (PscF‐SD; residues 42–332) was cloned into an appropriate expression vector, recombinantly produced in Escherichia coli BL21‐Gold(DE3) cells and purified via immobilized metal‐affinity and size‐exclusion chromatography. Purified PscF‐SD was screened for crystallization; the best crystal diffracted to a maximum resolution of 1.46 Å in space group P 2 1 2 1 2 1 . However, the data could not be phased using the only available NIT‐domain structure ( Klebsiella oxytoca NasR; PDB entry 4akk) as the search model. Therefore, a data set from a selenomethionine‐labelled protein crystal was also collected. The selenomethionine‐labelled protein crystal diffracted to a resolution of 2.46 Å in space group P 2 1 2 1 2 1 . These data will be used to attempt to solve the structure using the single‐wavelength anomalous diffraction technique. The structure is expected to provide insights into the ligand specificity of NIT domains and the role of NIT domains in chemotaxis.