Stabilization of the ternary complex EF-Tu.GTP.valyl-tRNA by ammonium salts

In a search for crystallizing conditions for the ternary complex EF-Tu.GTP.valyl-tRNA^val, the influence of various salts on its stability has been examined by measuring the rate of deacylation of the aminoacyl-tRNA in the complex. The most striking result is the general higher stability in solutions of ammonium salts and, in particular, the enhancement of this effect by sulphate and citrate. Thus sodium sulphate and citrate lead to destabilization of the complex, as expected from conventional considerations of adding salt, whereas the corresponding ammonium salts stabilize the complex as shown, for example, by an increase in the half-life of the valyl-tRNA^val in the complex from about 20 hours to at least 300 hours in the presence of 1.2 M ammonium sulphate. These results suggest that ammonium sulphate and ammonium citrate might be very suitable precipitants for crystallization studies of the ternary complex.     

Diagnostic of precipitant for biomacromolecule crystallization by quasi-elastic light-scattering

The translational diffusion coefficient D25,w of hen egg-white lysozyme and concanavalin A from the jack bean is measured in various precipitating agent solutions as a function of salt and protein concentration using quasi-elastic light-scattering. With some precipitants, in undersaturated protein solutions, a protein or salt concentration dependence of the diffusion coefficient of the scatters is observed. It can be correlated with the inability of the protein to crystallize in this precipitant once the solution is supersaturated. These variations of D25,w are interpreted in terms of non-specific interactions and/or aggregation that prevent the protein from making appropriate contacts to form a crystal. With other precipitants known to lead to crystallization, no significant variation of the diffusion coefficient with increasing concentration was observed, indicating that under such conditions up to saturation the proteins remain essentially monodisperse. Application of this technique to find crystallization conditions of other proteins is discussed.     

Tyr-426 of the Escherichia coli asparaginyl-tRNA synthetase, an amino acid in a C-terminal conserved motif, is involved in ATP binding

Sequence comparisons of the E. coli asparaginyl-tRNA synthetase (NRSEC) with aminocyl-tRNA synthetase sequences of class II enzymes show significant homologies with aspartyl- and lysyl-tRNA synthetases. Three conserved regions were found, one of which is located in the C-terminal part of the NRSEC sequence. Site-directed mutagenesis was performed in this conserved region. A single point mutation Tyr-426----Ser results in a 15-fold increase in the Km for ATP, while all the other kinetic parameters remain unchanged. The replacement of this Tyr-426 by a Phe does not affect the kinetic behaviour of the enzyme. These data indicate that Tyr-426 is part of the ATP binding site.     

The effect of acid mucopolysaccharides and acid mucopolysaccharide–proteins on fibril formation from collagen solutions

1. The effects of acid mucopolysaccharides and acid mucopolysaccharide-proteins on the size and rate of formation of fibril aggregates from collagen solutions in pH7.6 buffers were studied by turbidimetric and light-scattering methods. 2. Serum albumin, orosomucoid, methylated cellulose, chondroitin sulphate A and chondroitin sulphate C of molecular weight less than 20000, and hyaluronate of molecular weight less than 40000 did not influence rates of fibril formation. Chondroitin sulphate A, chondroitin sulphate C and hyaluronate of high molecular weight retarded the rate of fibril formation. This effect of high-molecular-weight chondroitin sulphate C decreased with increasing ionic strength. Heparin, though of low molecular weight (13000), was highly effective, as was also heparitin sulphate. The chondroitin sulphate-proteins of very high molecular weight were highly effective, despite the fact that for some preparations the component chondroitin sulphate chains had molecular weights much less than 20000. 3. Agents that had delayed fibril formation were also effective in producing an increase in degree of aggregation of fibrillar collagen, as indicated by dissymmetry changes observed in light-scattering experiments at low collagen concentrations. Methylated cellulose and heparin at 2.5mug./ml. were unusual in decreasing aggregation, but heparin at 0.25mug./ml. increased aggregation. Electron microscopy of gels showed fibrils and fibril aggregates with ;normal' collagen spacing and dimensions consistent with the light-scattering results. 4. The rates of electrical transport of agents and of solvent (electro-osmosis) through collagen gels indicated a contribution of molecular entanglement that increased with increase in molecular size of the agents. Electrostatic binding of heparin to collagen was noted. Binding to collagen during fibril formation was also found for heparitin sulphate and a chondroitin sulphate with extra sulphate groups. 5. Electrostatic binding of acid mucopolysaccharide-proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development. Excluded-volume (molecular-entanglement) effects may also be important. These factors operate simultaneously and interact mutually so that precise assessment of their relative importance is difficult.     

Asparaginyl-tRNA synthetase from Escherichia coli has significant sequence homologies with yeast aspartyl-tRNA synthetase

The Escherichia coli asnS gene codes for asparaginyl-tRNA synthetase (NRSEC). We have sequenced the asnS region, including 382 bp of the 5'-untranslated region, 1398 bp of the coding region and 280 bp of the 3'-untranslated region. The DNA-derived NRSEC amino acid (aa) sequence was confirmed by direct aa sequencing of the N-terminal parts of the native protein and of a 28-kDa internal fragment generated by trypsin digestion. The asnS gene product has been purified to homogeneity using three chromatographic steps. Sequence comparison of the deduced NRSEC sequence with all aminoacyl-tRNA synthetase sequences showed significant homologies with the yeast aspartyl-tRNA synthetase and weaker relationships with other aminoacyl-tRNA synthetases for aa with an XAX codon.     

Crystallization of Photobacterium leiognathi non-fluorescent flavoprotein, an unusual flavoprotein with limited sequence identity to bacterial luciferase.

Single crystals of the non-fluorescent flavoprotein (NFP) purified from Photobacterium leiognathi strain S1 have been grown from ammonium sulphate solutions using the hanging drop vapour diffusion technique. The crystals grow as thin (0.06 mm) plates and belong to the orthorhombic space group C222(1): a = 57.06(3) A, b = 92.41(6) A, c = 99.52(6) A. There is one NFP monomer per asymmetric unit and crystals diffract to 2.2 A spacings on film. A complete native data set to 2.5 A resolution has been collected on a San Diego Multiwire Detector system at the University of Alberta and a heavy-atom derivative search is presently in progress.     

Purification, crystallization and preliminary X-ray analysis of toxic shock syndrome toxin-1 from Staphylococcus aureus.

High yields of toxic shock syndrome toxin-1, from Staphylococcus aureus, have been purified (> 99%) using a novel, simple, two-step procedure involving dye ligand chromatography. Crystals suitable for X-ray diffraction work were obtained by vapour diffusion using ammonium sulphate and polyethylene glycol as precipitants. They belong to the orthorhombic space group C222(1), with unit cell dimensions a = 108.6 A, b = 177.6 A and c = 97.5 A, with three molecules per asymmetric unit. The crystals diffract to at least 2.5 A resolution and are suitable for three-dimensional X-ray structural analysis.     

Intermolecular complexation and phase separation in aqueous solutions of oppositely charged biopolymers

Turbidity measurements performed at 450nm were used to follow the process of complex formation, and phase separation in gelatin-agar aqueous solutions. Acid (Type-A) and alkali (Type-B) processed gelatin (polyampholyte) and agar (anionic polyelectrolyte) solutions, both having concentration of 0.1% (w/v) were mixed in various proportions, and the mixture was titrated (with 0.01 M HCl or NaOH) to initiate associative complexation that led to coacervation. The titration profiles clearly established observable transitions in terms of the solution pH corresponding to the first occurrence of turbidity (pH(C), formation of soluble complexes), and a point of turbidity maximum (pH(phi), formation of insoluble complexes). Decreasing the pH beyond pH(phi) drove the system towards precipitation. The values of pH(C) and pH(phi) characterized the initiation of the formation of intermolecular charge neutralized soluble aggregates, and the subsequent formation of microscopic coacervate droplets. These aggregates were characterized by dynamic light scattering. It was found that Type-A and -B gelatin samples formed soluble intermolecular complexes (and coacervates) with agar molecules through electrostatic and patch-binding interactions, respectively.     

Cloning,expression, crystallization,and preliminary X-ray analysis of recombinant mouse lipocalin-type prostaglandin D synthase,a somnogen-producing enzyme

Lipocalin-type prostaglandin D synthase is the key enzyme for the production of prostaglandin D(2), a potent endogenous somnogen, in the brain. We cloned, produced, and crystallized the native enzyme and selenomethionyl Cys(65)Ala mutants of the recombinant mouse protein by the hanging drop vapor-diffusion method with both malonate and citrate as precipitants. The native crystals obtained with malonate belong to orthorhombic space group P2(1)2(1)2(1) with lattice constants a = 46.2, b = 66.8, and c = 105.3 A. The selenomethionyl crystals obtained with citrate belong to orthorhombic space group C222(1) with lattice constants a = 45.5, b = 66.8, and c = 104.5 A. The native crystals diffracted beyond 2.1 A resolution.     

Solution studies of elongation factor Tu from the extreme halophile Halobacterium marismortui.

The activity, stability and structure in solution of polypeptide elongation factor hEF-Tu from Halobacterium marismortui have been investigated. The protein is stable in aqueous solutions only at high concentrations of NaCl, KCl or ammonium sulphate, whereas it is more active in exchanging GDP at lower salt concentrations. It is more active and stable at lower pH values than is non-halophilic EF-Tu. The structure in solution of the protein was determined by complementary density, ultracentrifugation, dynamic light-scattering and neutron-scattering measurements. The protein has large hydration interactions, similar to those of other halophilic proteins: 0.4 (+/- 0.1) g of water and 0.20 (+/- 0.05) g of KCl associated with 1 g of protein, with a water/KCl mass ratio always remaining close to 2. The kinetics of inactivation at low salt concentrations showed a stabilizing effect of NaCl when compared to KCl. At low salt concentration, inactivation, protein unfolding and aggregation were strongly correlated. The results suggest that the stabilization model proposed for halophilic malate dehydrogenase by Zaccai et al., involving extensive protein interactions with hydrated salt ions, is also valid for hEF-Tu.     

Crystallization and preliminary crystallographic studies of glucosamine-6-phosphate deaminase from Escherichia coli K12.

Hexameric glucosamine-6-phosphate deaminase from Escherichia coli has been crystallized isomorphously with both phosphate and ammonium sulphate as precipitants, over a wide pH range (6.0 to 9.0). The crystals belong to space group R32 and the cell parameters in the hexagonal setting are a = b = 125.9 A and c = 223.2 A. A complete native data set was collected to 2.1 A resolution. Self-rotation function studies suggest that the hexamers sit on the 3-fold axis and have point group symmetry 32, with a non-crystallographic dyad relating two monomers linked by an interchain disulfide bridge. A possible packing for the unit cell is proposed.     

Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus.

Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphoinositides into two parts, lipid-soluble diacylglycerol and the water-soluble phosphorylated inositol. Two crystal forms of Bacillus cereus PI-PLC have been obtained by the vapor diffusion technique. Hexagonal crystals were grown from solutions containing polyethylene glycol (PEG; 4,000 to 8,000 D). The space group of these hexagonal crystals is P6(1)22 (or the enantiomorphic space group P6(5)22), with cell constants a = b = 133 A, and c = 231 A. The crystals diffract to 2.8 A. The second crystalline form was grown from a two-phase PEG (600 D)-sodium citrate solution. The phase diagram and PI-PLC distribution between phases has been determined. The enzyme crystallizes from the PEG-rich phase. The crystals are orthorhombic with space group P2(1)2(1)2(1) (a = 45 A, b = 46 A, c = 160 A), and contain one PI-PLC monomer per asymmetric unit. The orthorhombic crystals diffract to 2.5 A. Both the hexagonal and orthorhombic forms are suitable for crystallographic studies.     

Recovery of canola meal proteins by precipitation

Recovery of rapeseed proteins from defatted canola meal by precipitation was investigated. The ability of different precipitating agents, such as sodium hexametaphosphate (HMP), carboxymethylcellulose (CMC), ammonium sulphate, and isoelectric precipitation using HCl, were evaluated based on the yield and mean size of protein aggregates. Almost 94% of dissolved protein was precipitated in the presence of 2.7M ammonium sulphate, while the largest mean protein particle size (32 mum) was obtained in the presence of HMP at pH 3.3.     

Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.

The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 degrees C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation.     

Irreversible gelation of thermally unfolded proteins: structural and mechanical properties of lysozyme aggregates

The formation of protein aggregates is important in many fields of life science and technology. The morphological and mechanical properties of protein solutions depend upon the molecular conformation and thermodynamic and environmental conditions. Non-native or unfolded proteins may be kinetically trapped into irreversible aggregates and undergo precipitation or gelation. Here, we study the thermal aggregation of lysozyme in neutral solutions. We characterise the irreversible unfolding of lysozyme by differential scanning calorimetry. The structural properties of aggregates and their mechanisms of formation with the eventual gelation are studied at high temperature by spectroscopic, rheological and scattering techniques. The experiments show that irreversible micron-sized aggregates are organised into larger clusters according to a classical mechanism of diffusion and coagulation, which leads to a percolative transition at high concentrations. At a smaller length scale, optical and atomic force microscopy images reveal the existence of compact aggregates, which are the origin of the aggregation irreversibility.     

Detection of polymeric forms of dihydroquercetin by optical absorption and light scattering

Two methods for the detection of long polymers in dihydroquercetin (DHQ) preparations has been developed. The first method is based on UV spectrophotometry. It was shown that the quantity of long polymers in aqueous solutions can be estimated by the ratio of the absorption bands at 328 and 290 nm, since the 328-nm band was attributed to the monomeric form of DHQ, whereas the 290-nm band was attributed to both the monomeric and polymeric forms. The second method is based on the high-sensitive measurement of light-scattering intensity in aqueous solutions of diluted DHQ preparations using a spectrofluorometer with crossed monochromators. It has been shown that the filtration of DHQ solutions through Millipore filters with a pore diameter of 0.05–0.45 microns makes it possible to nearly completely eliminate long polymers and their aggregates. Long polymers at high concentrations can aggregate. The longest polymers and their aggregates may be 0.1 mm in length, which leads to fluctuations in the light-scattering intensity on the second and minute time scale.     

Estimation of Water Diffusion Coefficients in Freeze-Concentrated Matrices of Sugar Solutions Using Molecular Dynamics: Correlation Between Estimated Diffusion Coefficients and Measured Ice-Crystal Recrystallization Rates

The diffusion coefficient of the water component in a freeze-concentrated matrix is a useful parameter for predicting and controlling the recrystallization rate of ice crystals in sugar solutions relevant to frozen desserts. Herein, application of molecular dynamics (MD) for estimating the water diffusion coefficient in a freeze-concentrated matrix of sugar solutions is described. Diffusion coefficients evaluated using MD with the optimized potentials for liquid simulations all atom force field and water models of three types (simple point charge, simple point charge extended, and transferable intermolecular potential-4 point) show a good positive linear relation with measured values, indicating that the MD methods used in this study are useful for predicting differences in water diffusion coefficients in a sugar freeze-concentrated matrix. Furthermore, similarly to measured values, the estimated diffusion coefficients show a good positive correlation with recrystallization rates of ice crystals, which suggests that MD is useful to predict differences in recrystallization rates of ice crystals in frozen sugar solutions.     

Time dependence of aggregation in crystallizing lysozyme solutions probed using NMR self-diffusion measurements

The time dependence of aggregation in supersaturated lysozyme solutions was studied using pulsed-gradient spin-echo NMR diffusion measurements as a function of lysozyme concentration at pH 6.0 and 298 K in the presence of 0.5 M NaCl. The measurements provide estimates of the weight-averaged diffusion coefficient of the monomeric to intermediate molecular weight lysozyme species present in the solution (very large aggregates and crystals are excluded from the average due to the NMR relaxation-weighting effects inherent in the method). The results show that the average molecular weight of the various lysozyme aggregates changed with sigmoidal kinetics and that these kinetics were strongly influenced by the initial lysozyme concentration. The visualization of the time dependence of the protein aggregation afforded by this method provides a deeper understanding of how the crystallizing conditions (especially the initial protein concentration) are related to the resulting crystals.     

Crystallization and preliminary X-ray investigation of bovine liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit.     

The oligomeric state, complex formation, and chaperoning activity of hsp70 and hsp80 of Neurospora crassa.

Molecular chaperones perform vital cellular functions under normal growth conditions and protect cells against stress-induced damage. The stress proteins Hsp70 and Hsp80 of Neurospora crassa were extracted from heat-shocked mycelium, purified to near homogeneity, and examined with respect to their oligomeric state, complex formation, and chaperoning properties. Their oligomeric state was assessed by dynamic light-scattering measurements, and both Hsp70 and Hsp80 were observed to form a range of soluble, high-molecular-mass protein aggregates. Direct interaction between Hsp70 and Hsp80 was studied by partial tryptic digestion and surface plasmon resonance (SPR). Hsp70 was immobilized on the sensor chip surface, and the binding of Hsp80 in solution was followed in real time. Proteolytic digestion revealed that Hsp70-Hsp80 complex formation results in conformational changes in both proteins. The data from SPR studies yielded an equilibrium dissociation constant, KD, of 8.5 x 10(-9) M. The chaperoning ability of Hsp70, Hsp80, and Hsp70-Hsp80 was monitored in vitro by the protection of citrate synthase from thermal aggregation. The binding of nucleotides modulates the oligomeric state, chaperoning function, and hetero-oligomeric complex formation of Hsp70 and Hsp80.