Crystallization and preliminary X-ray analysis of the recombinant dihaem cytochrome c (NapB) from Haemophilus influenzae.
The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase (Nap). Recombinant NapB was overproduced in Escherichia coli, purified to near-homogeneity and crystallized using the hanging-drop method. Thin quadrilateral plates were grown under various conditions but proved to be unsuitable for X-ray analysis. However, a single crystal was grown using 1.75 M ammonium sulfate in 0.1 M sodium acetate pH 5.5, from which a native data set could be collected to 1.8 A resolution using synchrotron radiation. Using the same conditions, further crystals were obtained by microseeding. The space group was determined to be P42(1)2, with unit-cell parameters a = 77.55, b = 77.55, c = 28.64 A and an unusually low solvent content of 16.5%, assuming there to be one molecule of NapB in the asymmetric unit. Analysis of the dissolved crystals indicated that partial proteolysis of the protein had occurred. Taking the molecular mass of the crystallized form ( approximately 8500 Da) into account, the solvent content was estimated to be 53%, with a V(M) value of 2.64 A(3) Da(-1).
Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, State University of Gent, B-9000 Gent, Belgium.
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Brigé
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Leys
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Van Beeumen
D003460:Crystallization; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry*; D006193:Haemophilus influenzae / Q000201:enzymology*; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D009565:Nitrate Reductases; D050901:Nitrate Reductase