Crystallization of butyrate kinase 2 from Thermotoga maritima mediated by vapor diffusion of acetic acid.
The sitting-drop method of crystallization uses the evaporation of water to increase the concentration of the protein and precipitant in the drop. The presence of other volatile components, such as acetic acid, can have a marked impact on crystallization. A member of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of proteins, isobutyrate kinase (Buk2) from Thermotoga maritima, was expressed in Escherichia coli with six histidine residues added to the C-terminus. The purified protein was crystallized in a sitting drop with a well solution consisting of 1.7-3.0 M sodium formate, with the pH of the well solution alone adjusted to 4.5 with acetic acid. Diffraction data collected at 100 K show that the crystals diffract to 3.1 A and belong to space group I422, with unit-cell parameters a = b = 198.12, c = 58.93 A. Both the crystal form and the results of dynamic light-scattering studies suggest that Buk2 is an octomer, the first to be identified in the ASKHA superfamily.
Diao
Jiasheng
J
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2054, USA.
Cooper
David R
DR
Sanders
David A
DA
Hasson
Miriam S
MS
D019342:Acetic Acid / Q000737:chemistry; D003460:Crystallization; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008970:Molecular Weight; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020124:Thermotoga maritima / Q000201:enzymology*; D014961:X-Ray Diffraction
D007202:Indicators and Reagents; D004247:DNA; D017851:Phosphotransferases (Carboxyl Group Acceptor); C480759:butyrate kinase 2, Thermotoga maritima; D019342:Acetic Acid