Purification, crystallization and preliminary X-ray data of the bacteriophage MS2

Single crystals of the bacteriophage MS2 have been produced by the vapour diffusion technique in the presence of 1.5% polyethylene glycol 6000 and 0.2 M-sodium phosphate buffer (pH 7.4). These are the first bacteriovirus crystals diffracting to high resolution. The crystal space group is C2 with the unit cell parameters a = 467.9 A, b = 289.5 A, c = 275.6 A and beta = 121.8 degrees. The asymmetric unit contains one half of the virion. The maximum resolution limit of the X-ray diffraction data obtained from these crystals was 2.9 A. The purification of the virus material was done by mild procedures exclusively and involved precipitation with polyethylene glycol 6000 and size exclusion chromatography on Sepharose CL-4B.     

Crystallization of yeast iso-2-cytochrome c using a novel hair seeding technique

A hair seeding technique has been developed to obtain diffraction quality crystals of yeast (Saccharomyces cerevisiae) iso-2-cytochrome c, a model for studies of protein folding and biological electron transfer reactions. Deep red crystals of this protein were obtained from 88 to 92% saturated solutions of ammonium sulfate containing 20 mg protein/ml, 0.1 M-sodium phoshate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to phosphate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to pH 6.0. Rapid crystal growth was observed, but only along the path of the seeding hair stroke. The space group is P4(3)2(1)2 (or P4(1)2(1)2) with a = b = 36.4 A, c = 137.8 A (1 A = 0.1 nm) and Z = 8. Crystals are stable in the X-ray beam for more than 10 days and diffract to at least 2.5 A resolution. The same hair seeding methodology has proven useful in obtaining crystals of specifically designed mutant iso-2 proteins and in other protein systems where consistent crystal growth had previously proven difficult to attain.     

Hybridization properties of nucleolar ribonucleic acid.

Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.     

Crystallization and preliminary diffraction data for iso-1-cytochrome c from yeast

Deep red crystals of the electron transfer protein, iso-1-cytochrome c from yeast (Saccharomyces cerevisiae), have been obtained from a 90% saturated solution of (NH4)2SO4 containing 2 mg protein/ml, 0.1 M-sodium phosphate and adjusted to pH 6.7. The space group is P4(1)2(1)2 (or P4(3)2(1)2) with a = b = 36.4 A, c = 136.8 A and Z = 8. Crystals are stable for at least ten days in the X-ray beam and diffract to better than 2.0 A resolution. Comparable and morphologically similar crystal forms of three iso-1-cytochrome c mutants at Phe87, a pivotal residue in the electron transport chain, have also been obtained.     

Crystallization of the DNA-binding Escherichia coli protein FIS

The specific DNA-binding protein FIS (factor for inversion stimulation), which stimulates site-specific DNA inversion by interaction with an enhancer sequence, was purified from an Escherichia coli strain overproducing the protein. FIS was crystallized at room temperature by microdialysis against 1.2 to 1.5 M-sodium/potassium phosphate containing 10 mM-Tris.HCl, 0.5 to 1 M-NaCl and 1 mM-NaN3 at pH 8.0 to 8.2. The crystals are stout prisms and suitable for X-ray diffraction study beyond 2.5 A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1). The unit cell has dimensions a = 47.57(4) A, b = 51.13(4) A, c = 79.83(6) A and contains one FIS dimer in the asymmetric unit.     

Human D-Phe-Pro-Arg-CH2-alpha-thrombin crystallization and diffraction data

Human alpha-thrombin, inhibited with the high-affinity irreversible inhibitor D-Phe-Pro-Arg-chloromethylketone, has been crystallized from polyethylene glycol 8000 solutions buffered with 0.1 M-sodium phosphate. The crystals are: orthorhombic, a = 67.9(1) A, b = 87.9(1) A, c = 61.0(1) A, space group P2(1)2(1)2(1) with four molecules per unit cell. This gives a protein fraction of 58% consistent with the excellent X-ray diffraction quality of the crystals. A mercury heavy-atom derivative is being prepared from a thioester analogue of D-Phe-Pro-Arg-CH2-alpha-thrombin in anticipation of a complete crystallographic structure determination.     

The investigation of substrate-induced changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenases by measurement of the kinetics and thermodynamics of subunit exchange.

1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.     

Characterization of the genome of the basidiomycete Schizophyllum commune

DNA of Schizophyllum commune was isolated both from mycelial cells and from protoplasts. Nuclear DNA was isolated after solubilization of the mitochondria with the detergent Nonidet. The G + C content of the nuclear DNA was 57%, calculated from its buoyant density (1.7165 g/ml) and from the Tm (77.4 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). The buoyant density of the ribosomal cistrons was 1.707 g/ml. DNA isolated from purified mitochondria had a very low G + C content: 22% (rho = 1.6845 g/ml, Tm = 61.8 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). Analysis of CsCl profiles and melting patterns suggested that mitochondrial DNA contains interspersed (A + T)-rich sequences. From reassociation analysis of sheared nuclear DNA the genome size of S. commune was determined to be 22.8 . 10(9) daltons. A small amount of DNA (0.5 . 10(9) daltons) bound to hydroxyapatite at zero time Cot. 7% of the genome (1.6 . 10(9) daltons) represented repetitive DNA.     

Calcium-dependent protein modulator of cyclic nucleotide phosphodiesterases from mouse epidermis.

1. A heat-stable modulator protein was partially purified from mouse epidermis. The protein stimulated modulator-depleted cyclic AMP phosphodiesterase from bovine brain in the presence of Ca2+. 2. DEAE-cellulose chromatography of epidermal extracts demonstrated the presence of two main phosphodiesterase activities that hydrolysed both cyclic AMP and cyclic GMP. A minor peak was eluted between 0.1 and 0.3 M-sodium acetate and a major peak was eluted between 0.3 and 0.45 M-sodium acetate. 3. Cyclic AMP phosphodiesterase activity eluted at low salt concentrations was markedly activated by the epidermal modulator protein in the presence of Ca2+. Storage of the enzyme led to a decrease in its sensitivity to the protein modulator. 4. Treatment of mouse skin with the tumour promoter 12-O-tetradecanoylphorbol 13-acetate, which leads to an increase in epidermal cyclic nucleotide phosphodiesterase activity, did not alter the amount of modulator present in soluble epidermal extracts. The tumour promoter decreased the amount of modulator extractable from particulate epidermal preparations with Triton X-100.     

Crystallization and preliminary X-ray crystallographic analysis of lipase from Pseudomonas cepacia.

Large crystals of lipase from Pseudomonas cepacia have been grown at room temperature from solutions containing 2-methyl-2,4-pentanediol and sodium citrate. They grow within two weeks to typical dimensions of 1.0 mm x 0.5 mm x 0.3 mm. The crystals belong to the monoclinic space group P2(1), with unit cell parameters a = 84.91 A, b = 47.33 A, c = 86.00 A, and beta = 116.09 degrees. And they diffract to about 1.6 A upon exposure to synchroton X-rays. X-ray data have been collected to 2.2 A Bragg spacing from a native crystal.     

Crystallization and preliminary crystallographic studies of the FAD domain of corn NADH: nitrate reductase.

Crystals of the flavin domain of corn nitrate reductase expressed in Escherichia coli have been obtained at room temperature, using sodium citrate as precipitant. The crystals diffract to at least 2.5 A resolution at a synchrotron radiation source. Precession photographs show that they belong to the rhombohedral space group R3 with unit cell dimensions a = b = 145.4 A, c = 47.5 A, alpha = beta = 90 degrees and gamma = 120 degrees. There is one subunit per asymmetric unit which gives a packing density of 3.2 A3/Da, indicating a high solvent content in these crystals.     

Autolysis of Clostridium acetobutylicum ATCC 824.

The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.     

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.     

Nuclear envelope of the seminal-vesicle epithelium.

The nuclear envelope of seminal-vesicle epithelium was isolated by a procedure involving enzymic digestion with deoxyribonuclease I, sonication in the presence of 0.34 M-sodium citrate, and centrifugation through sucrose density gradients. The mass of envelope DNA was only 0.8% of that of envelope protein, and by transmission electron microscopy the envelope was 98-99% pure. We showed that the envelope possess a protein kinase activity which is uninfluenced by cyclic nucleotides. Both lysine-rich histone and dephosphophosvitin as substrates gave a greater specific activity than did envelope protein itself. Optimum requirements with respect to Na+, Mg2+, pH and ATP were established for each substrate, and the influence of other factors on enzyme activity was investigated. Data, obtained mainly with the use of lysine-rich histone, are presented which indicate that nuclear envelope from intact and 96 h-castrated guinea pigs may have equal protein kinase activities and, in separate experiments, equal phosphoprotein phosphatase activities. Clarification of these initial observations must await identification of the natural substrates or the envelope's phosphorylation-dephosphorylation reactions.     

Citrate synthase from Thermus aquaticus: a thermostable bacterial enzyme with a five-membered inter-subunit ionic network

A bacterial thermostable citrate synthase has been analyzed to investigate the structural basis of its thermostability, and to compare such features with those previously identified in archaeal citrate synthases. The gene encoding the citrate synthase from Thermus aquaticus was identified from a gene library by screening with a PCR fragment amplified from genomic DNA using a primer based on the determined N-terminal amino acid sequence and a citrate synthase consensus primer. Apart from high sequence similarities with citrate synthase sequences within the Thermus/ Deinococcus group, the analyzed enzyme has highest similarities with the enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The recombinant enzyme is a dimer with high specific activity. Compared to its thermoactivity (T(opt)at 80 degrees C), the thermal stability of the enzyme is high, as judged from its T(m) (101 degrees C), and from irreversible thermal inactivation assays. Molecular modeling of the structure revealed an inter-subunit ion-pair network, comparable in size to the network found in the citrate synthase from P. furiosus; these networks are discussed in relation to the high thermal stability of these bacterial and archaeal enzymes.     

Crystal structure of an open conformation of citrate synthase from chicken heart at 2.8-A resolution

The X-ray structure of a new crystal form of chicken heart muscle citrate synthase, grown from solutions containing citrate and coenzyme A or L-malate and acetyl coenzyme A, has been determined by molecular replacement at 2.8-A resolution. The space group is P4(3) with a = 58.9 A and c = 259.2 A and contains an entire dimer of molecular weight 100,000 in the asymmetric unit. Both "closed" conformation chicken heart and "open" conformation pig heart citrate synthase models (Brookhaven Protein Data Bank designations 3CTS and 1CTS) were used in the molecular replacement solution, with crystallographic refinement being initiated with the latter. The conventional crystallographic R factor of the final refined model is 19.6% for the data between 6- and 2.8-A resolution. The model has an rms deviation from ideal values of 0.034 A for bond lengths and of 3.6 degrees for bond angles. The conformation of the enzyme is essentially identical with that of a previously determined "open" form of pig heart muscle citrate synthase which crystallizes in a different space group, with one monomer in the asymmetric unit, from either phosphate or citrate solution. The crystalline environment of each subunit of the chicken enzyme is different, yet the conformation is the same in each. The open conformation is therefore not an artifact of crystal packing or crystallization conditions and is not species dependent. Both "open" and "closed" crystal forms of the chicken heart enzyme grow from the same drop, showing that both conformations of the enzyme are present at equilibrium in solution containing reaction products or substrate analogues.     

Expression, purification, crystallization and preliminary crystallographic analysis of the human intracellular chloride channel protein CLIC4

The human chloride intracellular channel protein CLIC4 has been crystallized by the hanging-drop vapour-diffusion technique using trisodium citrate as the precipitant. The best crystals were obtained by the microseeding method. The crystals diffracted to 2.2 A resolution and were found to belong to space group P121, with unit-cell parameters a = 73.19, b =86.05, c = 73.38 A, beta = 112.99 degrees and three molecule per asymmetric unit.     

Interaction of a small heat shock protein of the fission yeast, Schizosaccharomyces pombe, with a denatured protein at elevated temperature

We have expressed, purified, and characterized one small heat shock protein of the fission yeast Schizosaccharomyces pombe, SpHsp16.0. SpHsp16.0 was able to protect citrate synthase from thermal aggregation at 45 degrees C with high efficiency. It existed as a hexadecameric globular oligomer near the physiological growth temperature. At elevated temperatures, the oligomer dissociated into small species, probably dimers. The dissociation was completely reversible, and the original oligomer reformed immediately after the temperature dropped. Large complexes of SpHsp16.0 and denatured citrate synthase were observed by size exclusion chromatography and electron microscopy following incubation at 45 degrees C and then cooling. However, such large complexes did not elute from the size exclusion column incubated at 45 degrees C. The denatured citrate synthase protected from aggregation was trapped by a GroEL trap mutant at 45 degrees C. These results suggest that the complex of SpHsp16.0 and denatured citrate synthase at elevated temperatures is in the transient state and has a hydrophobic nature. Analyses of the interaction between SpHsp16.0 and denatured citrate synthase by fluorescence cross-correlation spectrometry have also shown that the characteristics of SpHsp16.0-denatured citrate synthase complex at the elevated temperature are different from those of the large complex obtained after the shift to lowered temperatures.     

Purification and characterization of the reconstitutively active citrate carrier from maize mitochondria.

The citrate carrier from maize (Zea mays) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and hydroxyapatite/celite in the presence of cardiolipin. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31 kD. When reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal 5'-P-sensitive citrate/citrate exchange. It was purified 224-fold with a recovery of 50% and a protein yield of 0.22% with respect to the mitochondrial extract. In the reconstituted system the purified citrate carrier catalyzed a first-order reaction of citrate/citrate (0.065 min-1) or citrate/malate exchange (0.075 min-1). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate, cis-aconitate, isocitrate, L-malate, succinate, malonate, glutarate, alpha-ketoglutarate, oxaloacetate, and alpha-ketoadipate and was inhibited by pyridoxal 5'-P, phenylisothiocyanate, mersalyl, and p-hydroxymercuribenzoate (but not N-ethylmaleimide), 1,2, 3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10 degrees C and 35 degrees C; the half-saturation constant (Km) for citrate was 0.65 +/- 0.05 mM and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 +/- 1.0 micromol min-1 mg-1 protein at 25 degrees C.     

Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4

A thermotolerant yeast strain named Kluyveromyces marxianus IMB4 was used in a simultaneous saccharification and fermentation (SSF) process using Kanlow switchgrass as a feedstock. Switchgrass was pretreated using hydrothermolysis at 200 degrees C for 10 min. After pretreatment, insoluble solids were separated from the liquid prehydrolyzate by filtration and washed with deionized water to remove soluble sugars and inhibitors. Insoluble solids were then hydrolyzed using a commercial cellulase preparation and the released glucose was fermented to ethanol by K. marxianus IMB4 in an SSF process. SSF temperature was 37, 41, or 45 degrees C and pH was 4.8 or 5.5. SSF was conducted for 7 days. Results were compared with a control of Saccharomyces cerevisiae D(5)A at 37 degrees C and pH 4.8. Fermentation by IMB4 at 45 and 41 degrees C ceased after 3 and 4 days, respectively, when a pH 4.8 citrate buffer was used. Fermentation continued for all 7 days using IMB4 at 37 degrees C and the control. When pH 5.5 citrate buffer was used, fermentation ceased after 96 h using IMB4 at 45 degrees C, and ethanol yield was greater than when pH 4.8 citrate buffer was used (78% theoretical). Ethanol yield using IMB4 at 45 degrees C, pH 5.5 was greater than the control after 48, 72, and 96 h (P < 0.05).