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Improvements in neutron diffraction instrumentation are affording the opportunity to re-examine the structures of vertebrate hemoglobins and to interrogate proton and solvent position changes between the different quaternary states of the protein. For hemoglobins of unknown primary sequence, structural studies of cyanomethemoglobin (CNmetHb) are being used to help to resolve sequence ambiguity in the mass spectra. These studies have also provided additional structural evidence for the involvement of oxidized hemoglobin in the process of erythrocyte senescence. X-ray crystal studies of Tibetan snow leopard CNmetHb have shown that this protein crystallizes in the B state, a structure with a more open dyad, which possibly has relevance to RBC band 3 protein binding and erythrocyte senescence. R-state equine CNmetHb crystal studies elaborate the solvent differences in the switch and hinge region compared with a human deoxyhemoglobin T-state neutron structure. Lastly, comparison of histidine protonation between the T and R state should enumerate the Bohr-effect protons.

Room-temperature and 100 K X-ray and room-temperature neutron diffraction data have been measured from equine cyanomethemoglobin to 1.7 A resolution using a home source, to 1.6 A resolution on NE-CAT at the Advanced Photon Source and to 2.0 A resolution on the PCS at Los Alamos Neutron Science Center, respectively. The cyanomethemoglobin is in the R state and preliminary room-temperature electron and neutron scattering density maps clearly show the protonation states of potential Bohr groups. Interestingly, a water molecule that is in the vicinity of the heme group and coordinated to the distal histidine appears to be expelled from this site in the low-temperature structure.

Increasing the solubility of protein stock solutions to above that in a standard chromatography buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl) led to an increase in the number of crystallization conditions for ten globular proteins subjected to two crystal screens: the Index and Precipitant/Precipitant-Additive (P/PA) Screens. Solubility enhancement of protein stock solutions was achieved through screening and selection of buffer components to formulate an optimal buffer. Relative improvements in solubility were estimated through protection against the precipitation of protein by polyethylene glycol 8000. Proteins with limited solubility improvement in optimal buffer showed an enhancement in solubility on addition of glycerol. Maximum solubility was then determined by the concentration of optimized solutions until precipitate formed. The supernatant concentration then provided an estimate of the upper limit of protein solubility. This 'solubility' estimate is used to specify the initial concentration of the protein used in the screening experiments and is an important step in successful crystallization. Buffer optimization and establishment of initial protein concentration for crystal screening based on solubility estimates provides a methodology for improved crystal screening results.

The utility of a preliminary solubility screen has been assessed on ten test proteins. It is proposed that maximizing the protein solubility prior to crystal setups is likely to improve crystal growth. In crystallization setups, drops of a protein solution are mixed with various crystallization solutions which are then allowed to equilibrate. The protein solutions usually contain a salt and buffer which are present as a constant in all crystal screens. The propensity for crystallization, driven by three components of sparse-matrix screens, the buffers, salts and precipitating agents, could potentially be masked by the components of the protein solution. Ten test proteins were dissolved in a standard buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5) and in customized optimal buffers determined to maximize solubility. The proteins were then subjected to the Index (Hampton Research) 96-well sparse-matrix crystal screen and to a precipitant/precipitant-additive screen described here. Five of the ten proteins studied showed twofold to fourfold increases in the saturation level from standard to optimal buffer, two showed slight improvement and three showed a slight decrease. Microcrystals were obtained for all proteins and optimal buffer increased the appearance of crystals for eight of the ten proteins.

Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 angstroms, alpha = 71.2, beta = 68.1, gamma = 67.8 degrees and an estimated two molecules in the asymmetric unit, and diffract to 1.7 angstroms resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 angstroms, beta = 128.2 degrees and an estimated one molecule in the asymmetric unit, and diffract to 2.0 angstroms resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

Crystallization of protein and protein complexes is a multi-parametric problem that involves the investigation of a vast number of physical and chemical conditions. The buffers, salts and additives used to prepare the protein will be present in every crystallization condition. It is imperative that these conditions be defined prior to crystal screening since they will have a ubiquitous involvement in the crystal-growth experiments. This study involves the crystallization and preliminary analysis of the flap endonuclease-1 (FEN-1) DNA-repair enzyme from the crenarchaeal organism Aeropyrum pernix (Ape). Ape FEN-1 protein in a standard chromatography buffer had only a modest solubility and minimal success in crystallization trials. Using an ion/pH solubility screen, it was possible to dramatically increase the maximum solubility of the protein. The solubility-optimized protein produced large diffraction-quality crystals under multiple conditions in which the non-optimized protein produced only precipitate. Only minor adjustments of the conditions were required to produce single diffraction-quality crystals. The native Ape FEN-1 crystals diffract to 1.4 A resolution and belong to space group P6(1), with unit-cell parameters a = b = 92.8, c = 80.9 A, alpha = beta = 90, gamma = 120 degrees.