title: Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P.
abstract: The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified. The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6(1)22/P6(5)22, but the crystals diffract to only 3.4 A and are poorly reproducible. Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues. Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation. The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P2(1)2(1)2 or P2(1)2(1)2(1), with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 A, with diffraction to 2.4 A on a synchrotron source and with greatly improved reproducibility. Calculation of V(M) values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47%. A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, England.
authors.forename: S
authors.initials: S
authors.lastname: Bailey
authors.forename: S E
authors.initials: SE
authors.lastname: Sedelnikova
authors.forename: P
authors.initials: P
authors.lastname: Mesa
authors.forename: S
authors.initials: S
authors.lastname: Ayora
authors.forename: J C
authors.initials: JC
authors.lastname: Alonso
authors.forename: J B
authors.initials: JB
authors.lastname: Rafferty
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry*; D001435:Bacteriophages / Q000201:enzymology*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D002352:Carrier Proteins; C119221:G39P protein, Bacteriophage SPP1; D047908:Intracellular Signaling Peptides and Proteins; D017361:Viral Nonstructural Proteins