ESCHERICHIA-COLI P-II PROTEIN - PURIFICATION, CRYSTALLIZATION AND OLIGOMERIC STRUCTURE

The Escherichia coli signal transduction protein P-II, product of the glnB gene, was overproduced and purified. The predicted molecular weig ht of the protein based on the correct nucleotide sequence is 12,427 a nd is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylate d form of P-II with dimensions 0.2 x 0.2 x 0.3 mm were grown and analy sed by X-ray diffraction. The crystals belong to space group P6(3) wit h a = b = 61.6 Angstrom, c = 56.3 Angstrom and V-m of 2.5 for one subu nit in the asymmetric unit. A low-resolution electron density map show ed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight o f 35,000 +/- 1,000 for P-II in solution. This result is consistent wit h the native protein being a homotrimer.