The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding

Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.     

Crystallization of and preliminary X-ray data for the plasma retinol-binding protein

Crystals of the human and rabbit plasma retinol-binding proteins have been grown from solutions of polyethylene glycol 6000 and CdCl2. Two crystal forms have been observed for the human protein, while the rabbit protein has only crystallized in one form which is isomorphous with one of the human serum retinol-binding protein crystals. The crystals differ in their morphologies, but are both in space group P212121 and have similar unit cell sizes (a = 45.9, b = 53.3, c = 72.0 A and a = 45.7, b = 48.7, and c = 76.5 A). The crystals diffract to approximately 2.0 A resolution. In both cases there is 1 molecule/asymmetric unit.     

Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2.

The nanH genes of Vibrio cholerae and Salmonella typhimurium LT2 coding neuraminidase were cloned separately in Escherichia coli, and the expression products purified. Single crystals of the V. cholerae neuraminidase were obtained using the hanging drop vapour diffusion method with polyethylene glycol as precipitant at pH 7.2. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 71.9 A, b = 79.0 A, c = 165.7 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 2.5 A. Single crystals of the S. typhimurium neuraminidase were obtained by hanging drop with potassium phosphate as precipitant at pH 7.2. The crystals also belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 47.4 A, b = 82.8 A, c = 92.4 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 1.8 A.     

Crystallization and preliminary X-ray investigation of a sarcoplasmic calcium-binding protein from amphioxus

Crystals of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group C222(1), with unit cell axes a = 59.6(1) A, b = 81.3(1) A and c = 82.4(1) A. There is one molecule in the asymmetric unit. The crystals diffract beyond 2.5 A and show less than 20% decline in diffraction intensities after a three day exposure to X-rays from a laboratory rotating anode source.     

Crystallization of rat intestinal fatty acid binding protein. Preliminary X-ray data obtained from protein expressed in Escherichia coli

Rat intestinal fatty acid binding protein has been expressed in Escherichia coli, purified with bound long chain fatty acids and crystals grown from solutions of polyethylene glycol 4000. The crystals are monoclinic, space group P2(1), a = 3638 A, b = 57.2 A, c = 31.9 A, and beta = 113.9 degrees. Each unit cell contains two monomers of this 132-residue, 15.1-kDa polypeptide. The crystals are remarkably resistant to x-ray damage. X-ray diffraction data have been observed to 2.0 A resolution. Platinum chloride was used to generate a potential isomorphous heavy atom derivative.     

Crystallization of a high potential iron-sulfur protein from the halophilic phototrophic bacterium Ectothiorhodospira halophila

Crystals of the high-potential iron-sulfur protein from Ectothiorhodospira halophila strain BN 9626 have been grown from 3.4 to 3.5 M ammonium sulfate solutions at pH 7.5. The crystals belong to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. There are 2 molecules/asymmetric unit. The crystals diffract to at least 1.8 A, are stable in the x-ray beam, and are suitable for a high resolution x-ray crystallographic analysis.     

Crystallization and preliminary diffraction studies of 5 S rRNA from the thermophilic bacterium Thermus flavus

Crystals of purified 5 S rRNA from Thermus flavus have been obtained. The crystals diffract up to 8 A resolution, using synchrotron radiation, and have the monoclinic space-group C2. The unit cell has the dimensions a = 190 A, b = 110 A, c = 138 A and beta = 117 degrees. The cell volume suggests the presence of four 5 S rRNA molecules per asymmetric unit.     

Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF.

Crystals of glutathione-S-transferase (GST)-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X-ray crystallographic analysis revealed that crystals of the GST-fused protein belong to space group P6(1)22 or P6(5)22 with unit cell dimensions a = b = 140.4 A, c = 93.5 A and gamma = 120 degrees, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 A resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA-binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well-ordered large crystals were unsuccessful. A possible application of GST-fusion proteins for small target proteins or domains to obtain crystals suitable for X-ray structure determination is proposed.     

Crystallization of the A alpha subunit of protein phosphatase 2A.

The A alpha subunit of human protein phosphatase 2A forms crystals in space group P2(1) with cell dimensions a = 104.0, b = 174.9, c = 168.2 A, and beta angle = 90.2 degrees. At cryogenic temperatures, the crystals diffracted to a resolution limit of approximately 3.0 A. Based on the unit cell dimensions and a calculated molecular mass of 65,277 Da, the Matthews coefficient suggests eight molecules per asymmetric unit. Two native data sets were collected to a nominal resolution of 3.0 A and merged to provide a set that is 93% complete, with Rsym of 9.9%.     

Characterization and preliminary crystallographic data on the VL-related fragment of the human kI Bence Jones protein Wat

A “naturally occurring” human κI V_L dimer, designated Wat, has been isolated and crystallized. Protein Wat consists of two non-covalently bound monomers, each having a molecular weight of ~ 11,500. The monomer subunit is composed of an entire variable region light chain (V_L) domain closely homologous to that of the κI Bence Jones protein Roy (Hilschmann &; Craig, 1965) as evidenced from amino acid composition, tryptic peptide map, and sequence analysis. Immunochemical studies substantiated that protein Wat is of the κ chain subgroup κI and lacks the isotypic and allotypic antigenic determinants associated with the κ constant region light chain domain. Two types of crystals of V_L dimer Wat were obtained from ammonium sulfate or polyethylene glycol solutions. The type I crystals have unit cell dimensions of $\text{a = b = 82.6}\text{A}\text{?}\text{, c = 60.3}\text{A}\text{?}$, and the space group is hexagonal P6₂ or P6₄. The asymmetric unit consists of one V_L dimer; the fractional volume of unit cell occupied by solvent is 0.51. The unit cell dimensions of the type II crystals are $\text{a = b = 1,08.3}\text{A}\text{?}\text{, c = 108.8}\text{A}\text{?}$; the space group is hexagonal P6₁22 or P6₅22. Three variable domains constitute the asymmetric unit of the type II crystals; the fractional value of the solvent (0.52) is compatible with the value obtained for the type I crystals.     

Crystallization and preliminary X-ray diffraction studies of a 40 kDa calcium binding protein specifically expressed in plasmodia of Physarum polycephalum.

A calcium binding protein with a molecular mass of 40 kDa (CBP40), the gene product of plasmodial-specific LAV1-2 of Physarum polycephalum, was crystallized in the presence of EDTA. The crystals diffracted X-rays up to a resolution of 3.0 A. They belonged to the trigonal space group, P3221 (or P3121), with unit cell dimensions of a = b = 64.4 A and c = 207.2 A. Ca2+-bound crystals were obtained by soaking in a CaCl2 solution, which gave diffraction data of similar quality. The Ca2+-soaked crystals belonged to the same space group as those crystallized in the presence of EDTA with unit cell dimensions of a = b = 64.4 A and c = 209.4 A.     

Single crystals of large ribosomal particles from Halobacterium marismortui diffract to 6 A

Large, well-ordered three-dimensional crystals of 50 S ribosomal subunits from Halobacterium marismortui have been obtained by seeding. The crystals have been characterized with synchrotron X-ray radiation as monoclinic, space group P2(1), with unit cell dimensions of a = 182(+/- 5) A, b = 584(+/- 10) A, c = 186(+/- 5) A, beta = 109 degrees. At 4 degrees C, the crystals (0.6 mm X 0.6 mm X 0.1 mm) diffract to 6 A resolution and are stable in the synchrotron beam for several hours. Compact packing is reflected from the crystallographic unit cell parameters and from electron micrographs of positively stained thin sections of embedded crystals.     

Crystallization of the Bacillus subtilis histidine-containing phosphocarrier protein HPr and of some of its site-directed mutants

The histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been crystallized. Two of the site-directed mutants aimed at probing function produce crystals suitable for X-ray studies. The mutant in which His15 is substituted by an alanyl residue crystallizes from ammonium sulfate solution in space group P3(1)21 or P3(2)21, with unit cell dimensions: a = b = 47.3 A; c = 61.5 A. These crystals diffract to at least 1.8 A resolution. The mutant in which Ser46 is substituted by an aspartyl residue crystallizes from polyethylene glycol 4000 solution in space group P2(1), with unit cell dimensions: a = 49.4 A; b = 25.6 A; c = 60.3 A; beta = 109 degrees. These crystals diffract to at least 2.0 A resolution.     

Crystallization and preliminary X-ray crystallographic properties of Hsc20, a J-motif co-chaperone protein from Escherichia coli.

Hsc20 is a 20-kDa auxiliary protein that functions with the molecular chaperone Hsc66 in Escherichia coli. Crystals of Hsc20 suitable for X-ray diffraction analysis were grown using the hanging drop vapor diffusion technique in polyethylene glycol 400 containing dioxane as an additive to slow growth. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions a = 125.4 A, b = 71.9 A, c = 68.8 A, and beta = 97.0 degrees. The crystals diffract to a minimum d-spacing of approximately 2.5 A resolution, and a native data set was collected to 2.7 A. The results of a self-rotation function analysis revealed threefold symmetry, suggesting three molecules of Hsc20 in the asymmetric unit and, hence, 12 molecules in the unit cell; this corresponds to a Vm value of 2.6 A3/Da and a solvent content of approximately 53% in the crystals. Structure determination by isomorphous replacement is in progress.     

Crystallization and preliminary analysis of crystals of high potential iron-sulfur protein from Rhodospirillum tenue

Large single crystals of the high potential iron-sulfur protein isolated from Rhodospirillum tenue strain 3761 have been obtained. They belong to the space group P2(1) with unit cell dimensions of a = 36.7 A, b = 52.6 A, c = 27.6 A, and beta = 90.8 degrees. There are two molecules in the asymmetric unit. Based on oscillation photographs, the crystals diffract to at least 1.6 A resolution. They are stable in the x-ray beam and appear suitable for a high resolution x-ray structure analysis.     

X-ray studies on triclinic crystals of fatty acid binding protein. Examples of an extremely X-ray-resistant protein

Fatty acid binding protein (pI 7.0) from bovine liver cytosol was crystallized using polyethylene glycol 4000 and 6000 as precipitating agents. The crystals are triclinic, space group P1. One molecule of 14 kDa occupies the unit cell with constants a = 33.5 A, b = 39.4 A, c = 30.6 A, alpha = 113.6 degrees, beta = 113.8 degrees, gamma = 88.8 degrees. Crystal diffraction extends to at least 2.25 A resolution and the crystals are stable in the X-ray beam for more than 450 h. One native data set to 2.5 A resolution has been collected.     

Crystallization of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli

Single crystals of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli have been grown. This represents one of the first Fab-protein antigen complexes in which the same Fab fragment has previously been crystallized in the uncomplexed state and the structure solved (Prasad, L., Vandonselaar, M., Lee, J. S., and Delbaere, L. T. J. (1988) J. Biol. Chem. 263, 2571-2574). Single crystals up to 0.25 x 0.50 x 0.05 mm in size were grown by the technique of washing and reseeding. The space group is C2, with unit cell dimensions a = 130.0, b = 68.1, and c = 77.6 A; beta = 97.3 degrees; and Z = 4. There is one Fab-histidine-containing protein complex/asymmetric unit, and the solvent content is estimated to be 57%.     

Crystallization and characterization of the high molecular weight form of nerve growth factor (7 S NGF)

A high molecular weight form of nerve growth factor (7 S NGF) has been crystallized in two crystal forms from polyethylene glycol 4000 by the vapour diffusion technique. The orthorhombic form A belongs to the space group P2(1)2(1)2(1) and has cell dimensions of a = 95.6, b = 96.5 and c = 147.0 A. With synchrotron X-ray radiation, these crystals diffract to 2.8 A resolution. They contain an intact 7 S NGF complex in the asymmetric unit. The tetragonal form B, which grows at similar conditions to the A form, belongs to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = 97.4, b = 97.4 and c = 308.3 A. These crystals diffract to 3.6 A resolution and contain one 7 S complex per asymmetric unit. Native X-ray data have been collected to 3.3 A for the A form and to 5.0 A for the B form, both using synchrotron radiation.     

Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli: structure in projection at 40 A resolution.

SecA is a single-chain, membrane-associated polypeptide (102 kDa) which functions as an essential component of the protein export machinery of Escherichia coli. SecA has been crystallized from ammonium sulfate as small, three-dimensional bipyramidal crystals (0.1 x 0.1 x 0.05 mm). These crystals did not demonstrate detectable diffraction of X-rays from rotating anode sources. For study by electron microscopy, individual crystals were cross-linked in glutaraldehyde and OsO4 solutions, dehydrated, embedded in epoxy resin, and sectioned normal to crystallographic axial directions inferred from the external morphology of the crystals. Fourier transformation of processed images of untilted thin sections stained with uranyl acetate and lead citrate show reflections extending to 31 A resolution. Diffraction data and reconstructed images of the projected density of the unit cell contents indicate that the bipyramidal SecA crystals belong to orthorhombic space group C222(1) with unit cell dimensions a = 414 A, b = 381 A, and c = 243 A. Filtered images and density maps of mutually orthogonal projections of the unit cell contents are consistent with a three-dimensional model in which the asymmetric unit contains eight SecA monomers. The large unit cell dimensions and packing of protein monomers suggest that SecA is crystallizing as an oligomer of either dimers or tetramers.     

Crystallization of recA protein from Proteus mirabilis

The recA protein from Proteus mirabilis, which is homologous to the Escherichia coli protein, forms crystals in the orthorhombic space group P2(1)2(1)2(1). There are two 38,000 molecular weight subunits in the asymmetric unit and the unit cell dimensions are a = 57.5, b = 127.0 and c = 157.0 A.