Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa PelD.
The production of the PEL polysaccharide in Pseudomonas aeruginosa requires the binding of bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) to the cytoplasmic GGDEF domain of the inner membrane protein PelD. Here, the overexpression, purification and crystallization of a soluble construct of PelD that encompasses the GGDEF domain and a predicted GAF domain is reported. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as flat plates, with unit-cell parameters a = 88.3, b = 114.0, c = 61.9 Å, α = β = γ = 90.0°. The PelD crystals exhibited the symmetry of space group P2(1)2(1)2 and diffracted to a minimum d-spacing of 2.2 Å. On the basis of the Matthews coefficient (V(M) = 2.29 Å(3) Da(-1)), it was estimated that two molecules are present in the asymmetric unit.
Research Institute, Hospital for Sick Children, Toronto, ON, Canada.
Lindsey S
LS
Marmont
John C
JC
Whitney
Howard
H
Robinson
Kelly M
KM
Colvin
Matthew R
MR
Parsek
P Lynne
PL
Howell
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D008565:Membrane Proteins