Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P.
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.
Bailey
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Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, England.
Sedelnikova
S E
SE
Mesa
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P
Ayora
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S
Alonso
J C
JC
Rafferty
J B
JB
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
D002352:Carrier Proteins; C119221:G39P protein, Bacteriophage SPP1; D047908:Intracellular Signaling Peptides and Proteins; D017361:Viral Nonstructural Proteins