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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 m m NaCl) or in the presence of bivalent cations in the millimolar range (>= 5 m m 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 m m, 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 m m) probably promotes complete association of gp6 into 13-mer rings with a compact conformation

Abstract 1175 Adoptive transfer of T cells with specificity for one or several viruses is a promising strategy for the treatment or prevention of multiple opportunistic infections after allogenic stem cell transplantation. Here we have established a protocol for the generation of CMV, EBV and adenovirus (tri-virus) specific T cells. Virus-specific CD4+ and CD8+ T cells are stimulated in vitro using pools of peptides and subsequently magnetically enriched by virtue of their IFN-g secretion. The peptide pools are composed of synthetic peptides of mainly 15 amino acid length with 11 amino acid overlap covering the complete protein sequence of a viral antigen. We have analyzed the efficiency of short-term in vitro restimulation of cytomegalovirus (CMV)-specific CD4+ and CD8+ effector/memory T cells from CMV-infected healthy donors by CMV pp65 and IE-1 peptide pools as compared to single MHC class I restricted immunodominant pp65 and IE-1 9-mer peptides and CMV pp65 and IE-1 recombinant proteins based on flow cytometric analysis of the IFN-g response. The results show that peptide pools as well as recombinant proteins are useful for efficient activation of CD4+ T helper cells. In contrast, efficient stimulation of CD8+ T cells is achieved only using either the overlapping 15-mer peptide pools or the immunodominant 9-mer peptides. To test the usability of peptide pools to generate multi-virus-specific CD4+ and CD8+ T cells for adoptive immunotherapy we stimulated PBMC of several healthy donors with a combination of 5 peptide pools selected from CMV pp65, CMV IE-1, adenovirus hexon, EBV EBNA-1 and EBV BZLF-1 for 4 hours. Concomitant addition of 4 peptide pools to a single PBMC sample might decrease the activation efficiency for each peptide pool due to competition of peptides for MHC binding. Thus, as a control, we have divided PBMC into 4 aliquots, incubated each aliquot with a single peptide pool for 2 hours, and recombined the aliquots for T cell stimulation for another 4 hours. Afterwards, we used the IFN-g secretion assay technology to magnetically select for antigen-activated IFN-γ-secreting CD4+ and CD8+ T cells to a purity of > 90%. Antigen specificity and functionality of the enriched T cell population was examined after expansion. Within 9 – 14 days, T cells expanded between 4 and 745 fold. In addition, 21–53% of CD4+ T cells and 53–87% of CD8+ T cells re-expressed IFN-γ upon re-stimulation with the mixture of 4 peptide pools, confirming the high antigen specificity and functionality of the expanded T cells. Enrichment and expansions of T cells works equally well independent on whether the T cells are specific for CMV pp65, CMV IE-1, adenovirus hexon, EBV EBNA-1 or EBV BZLF-1. This can be deduced from the fact that the relative frequencies of T cells specific for each single antigen are about the same before and after the coenrichment and coexpansion process. For both approaches, the concomitant and separate antigen loading, we found comparable results with respect to recovery and purity of enriched IFN-γ-secreting T cells, expansion rate and antigen specificity of the expanded T cells. In summary, our results show that the combined usage of several peptide pools enables simultaneous and efficient activation of CMV, EBV and adenovirus (tri-virus) specific CD4+ and CD8+ T cells. Activated tri-virus specific T cells can be coenriched using the IFN-g secretion assay technology and used either directly or after in vitro coexpansion for adoptive immunotherapy. Disclosures: Richter: Miltenyi Biotec GmbH: Employment. Foerster:Miltenyi Biotec GmbH: Employment. Lasmanowicz:Miltenyi Biotec GmbH: Employment. Brauns:Miltenyi Biotec GmbH: Employment. Kramer:Miltenyi Biotec GmbH: Employment. Jekow:Miltenyi Biotec GmbH: Employment. Rönspeck:Miltenyi Biotec GmbH: Employment. Assenmacher:m: Employment. Schmitz:Miltenyi Biotec GmbH: Employment.

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.

Monomers of Photosystem I (PS I) from the thermophilic cyanobacterium Synechococcus elongatus were prepared and characterized. Using both the zwitterionic sulfobetain 12 and the nonionic β-dodecyl maltoside as detergents, PS I was extracted in trimeric form from the thylakoid membrane. The trimers dissociate into monomers during gel-filtration, apparently due to a change in the osmotic pressure. The PS I monomer prepared in β-dodecyl maltoside exhibits the same subunit composition as the trimeric PS I. The preparation in sulfobetain 12, however, leads to the removal of the two hydrophobic subunits K and L from the monomer. Further treatment of this sample with sulfobetain 12 detaches the subunits F, J and M, associated with the disappearance of a 706 nun band in the absorption spectrum. Both the PS I monomer with complete subunit composition and the monomer lacking subunits K and L were crystallized using batch and vapor diffusion techniques with poly(ethylene glycol) as the precipitating agent.

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.

Photosystem I monomers from wildtype cells of Synechocystis PCC 6803 and from a mu­ tant deficient in the psaL gene were crystallized. PsaL encodes for the hydrophobic subunit L, which has been proposed to constitute the trimerization domain in the PS I trimer. The absence of subunit L facilitated crystallization of the PS I monomer. The unit cell dimensions and the space group for the crystals from this preparation could be determined to be a = b = 132 Å , c -525 Å, α = β = 90°, y = 120°, the space group is P61 or P65. The results show the potential of using specifically designed deletion mutants of an integral membrane protein for the systematic improvement of crystal structure data.