Effect of the ionic environment on the molecular structure of bacteriophage SPP1 portal protein.

Bacteriophage SPP1 portal protein is a large cyclical homo-oligomer composed of 13 subunits. The solution structure and assembly behavior of this protein with high-point rotational symmetry was characterized. The purified protein was present as a monodisperse population of 13-mers, named gp6H, at univalent salt concentrations in the hundred millimolar range (>/= 250 mM NaCl) or in the presence of bivalent cations in the millimolar range (>/= 5 mM MgCl2). Gp6H had a slightly higher sedimentation coefficient, a smaller shape-dependent frictional ratio, and a higher rate of intersubunit cross-linking in the presence of magnesium than in its absence. In the absence of bivalent cations and at univalent salt concentrations below 250 mM, the 13-mer molecules dissociated partially into stable monomers, named gp6L. The monomer had a somewhat different shape from the subunit present in the 13-mer, but maintained a defined tertiary structure. The association-dissociation equilibrium was mainly between the monomer and the 13-mer with a minor population of intermediate oligomers. Their interconversion was strongly influenced by the ionic environment. Under physiological conditions, the concentration of Mg2+ found in the Bacillus subtilis cytoplasm (10-50 mM) probably promotes complete association of gp6 into 13-mer rings with a compact conformation.

Proteolytic cleavage of gram-positive beta recombinase is required for crystallization.

Beta recombinase, a DNA resolvase-invertase, catalyzes in the presence of a chromatin-associated protein such as Hbsu, DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented target (six) sites. Single crystals of the beta recombinase from plasmid pSM19035 were obtained using the vapor diffusion technique with ammonium phosphate as the precipitating agent. The crystals diffracted X-rays to a maximum resolution of 2.5A. Due to proteolytic degradation during the crystallization experiment, the crystals contain only the N-terminal catalytic domain of beta recombinase corresponding to about 60% of the molecular mass of the initially assayed native protein. The proteolytic removal of the C-terminal DNA-binding domain demonstrated that protein modification can be essential to provide material suitable for X-ray analysis.

Crystallization and preliminary X-ray crystallographic studies of the 13-fold symmetric portal protein of bacteriophage SPP1.

Portal proteins are cyclical oligomers which play essential roles in bacteriophage pro-capsid formation, DNA packaging, and in connector formation. Bacteriophage SPP1 portal protein (gp6) is a turbine-like molecule with 13-fold symmetry [Dube et al. (1993) EMBO J. 12, 1303-1309]. The purified protein was crystallized with polyethylene glycol 400 as the precipitating agent using the vapor-diffusion method. Salt conditions were selected based on the properties of gp6 in different ionic environments. X-ray diffraction data up to a resolution of 7.85 A were measured from frozen crystals with orthorhombic space group C2221 and cell dimensions a = 180.5 (5), b = 223.5 (5), c = 417 (1) A. The asymmetric unit contains one tridecameric portal protein with 57.3 kDa subunits. The self-rotation searches confirm the 13-fold symmetry of the crystallized protein.

Spectroscopic characterization of PS I core complexes from thermophilic Synechococcus sp. Identical reoxidation kinetics of A1- before and after removal of the iron-sulfur-clusters FA and FB.

Monomeric and trimeric PS I complexes missing the three stromal subunits E,C and D (termed PS I core complexes) were prepared from the thermophilic cyanobacterium Synechococcus sp. by incubation with urea. The subunits E,C and D are sequentially removed. In the monomeric PS I the subunit C is removed with a half life of approx. 5 min. This is about eight times faster than in the trimeric PS I complex. In parallel with the removal of the FA/B containing subunit C the reduction kinetics of P700+ changed from a half life of about 25 ms to about 750 microseconds. The partner of P700+ in the 750 microseconds charge recombination was identified to be FX by the difference spectrum of this phase. There are some minor differences in the spectra of trimeric and monomeric PS I core complexes. At 77K the forward electron transfer from A1- to FX is blocked in the major fraction of the PS I core complexes and P700+ A1- recombines with a half life of about 220 microseconds. In the remaining fraction P700+FX- is formed and decays with a half life of approx. 10 ms at 77 K. The kinetics of the forward electron transfer from A1- to the iron-sulfur-clusters was measured in the native PS I and the corresponding core complexes. The reoxidation kinetics of A1- are identical in both cases (t1/2 = 180 ns). We conclude that FX is an obligatory intermediate in the normal forward electron transfer.