Purification and crystallization of human cathepsin D.

The two-chain form of human cathepsin D was purified from human spleen with a method utilizing an ion exchange chromatography step prior to the pepstatin affinity column normally used to purify aspartic proteases. The protein was crystallized from 21% polyethylene glycol 8000 at pH 4.0 using the hanging drop vapour diffusion method. Small crystals were used as seeds to grow crystals suitable for X-ray data collection. The crystals diffract to a resolution of 3.2 A and have space group P2(1)2(1)2(1) with unit cell dimensions a = 59.9 A, b = 99.6 A, c = 133.6 A. There are two molecules in the asymmetric unit.     

Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae.

Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.     

Crystallization and preliminary X-ray characterization of a soybean seed lipoxygenase

An isoenzyme of soybean (Glycine max L. Merrill cv. Provar) lipoxygenase (EC 1.13.11.12) has been crystallized using the vapor diffusion method. Crystals were grown from solutions of the protein (7 mg/ml) using 10 to 20% (w/v) polyethylene glycol 8000 in citrate/phosphate buffer (pH 5.7) containing 0.5% (w/v) n-octyl-beta-D-glucopyranoside. The crystals reached maximum dimensions of 0.3 mm x 0.2 mm x greater than 2 mm. The enzyme crystallized in space group C222(1) with unit cell dimensions a = 246 A, b = 193 A and c = 75 A. A calculated Vm value of 2.35 A3/dalton was obtained assuming two molecules per asymmetric unit. The density of the crystals was found to be 1.16 g/ml, which confirmed the presence of two molecules per asymmetric unit and indicated a solvent content of 47.5%.     

Crystal structure of alginate lyase A1-III from Sphingomonas species A1 at 1.78 A resolution.

The three-dimensional structure of alginate lyase A1-III (ALYIII) from a Sphingomonas species A1 was determined by X-ray crystallography. The enzyme was crystallized by the hanging-drop vapour-diffusion method in the presence of 49% ammonium sulfate at 20 degrees C. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions of a=49.18 A, b=93.08 A, c=82.10 A and beta=104.12 degrees. There was one molecule of alginate lyase in the asymmetric unit of the crystal. The diffraction data up to 1. 71 A were collected with Rsymof 5.0%. The crystal structure of ALYIII was solved by the multiple isomorphous replacement method and refined at 1.78 A resolution using X-PLOR with a final R -factor of 18.0% for 10.0 to 1.78 A resolution data. The refined model of ALYIII contained 351 amino acid residues, 299 water molecules and two sulfate ions. The three-dimensional structure of ALYIII was abundant in helices and had a deep tunnel-like cleft in a novel (alpha6/alpha5)-barrel structure, which was similar to the (alpha6/alpha6)-barrel found in glucoamylase and cellulase. This structure presented the possibility that alginate molecules might penetrate into the cleft to interact with the catalytic site of ALYIII.     

Crystal structures of Aspergillus oryzae aspartic proteinase and its complex with an inhibitor pepstatin at 1.9A resolution

The X-ray structures of Aspergillus oryzae aspartic proteinase (AOAP) and its complex with inhibitor pepstatin have been determined at 1.9A resolution. AOAP was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=49.4A, b=79.4A, and c=93.6A. By the soaking of pepstatin, crystals are transformed into a monoclinic system with the space group C2 and cell dimensions of a=106.8A, b=38.6A, c=78.7A, and beta=120.3 degrees. The structures of AOAP and AOAP/pepstatin complex were refined to an R-factor of 0.177 (R(free)=0.213) and of 0.185 (0.221), respectively. AOAP has a crescent-shaped structure with two lobes (N-lobe and C-lobe) and the deep active site cleft is constructed between them. At the center of the active site cleft, two Asp residues (Asp33 and Asp214) form the active dyad with a hydrogen bonding solvent molecule between them. Pepstatin binds to the active site cleft via hydrogen bonds and hydrophobic interactions with the enzyme. The structures of AOAP and AOAP/pepstatin complex including interactions between the enzyme and pepstatin are very similar to those of other structure-solved aspartic proteinases and their complexes with pepstatin. Generally, aspartic proteinases cleave a peptide bond between hydrophobic amino acid residues, but AOAP can also recognize the Lys/Arg residue as well as hydrophobic amino acid residues, leading to the activation of trypsinogen and chymotrypsinogen. The X-ray structure of AOAP/pepstatin complex and preliminary modeling show two possible sites of recognition for the positively charged groups of Lys/Arg residues around the active site of AOAP.     

Thermodynamical model for insertion and aggregate binding of caffeine to the homopolymer poly(riboadenylate) and model choice by data analysis

This paper describes the model used to estimate the parameters of caffeine-poly(riboadenylate) (poly(A)) interactions from corresponding 1H-NMR measurements. The model of insertion and aggregate binding describes the non-cooperative insertion of a molecule C into an interspace between two monomers of a homopolymer in competition with aggregate binding. It contains two binding constants, K1 for insertion and K2 for the interaction of monomeric A units of the polymer with C molecules in bound aggregates, and two cooperativity parameters, Kcc for stacking of C molecules within aggregates and tau which is thought to be due to conformational adaptation of the polymer to those bound aggregates which cover more than one A unit. In contrast to other models, the size of a binding site (within the aggregates) is less than one monomeric unit, with n denoting the maximum number of C molecules per A unit in bound aggregates. The model is developed for general n by means of the method of sequence-generating functions. For n = 2 and n = 3, the correctness of the model treatment was checked by the matrix method. The model is applicable to the binding of aggregates to homopolymers, which are flexible enough to fit their structure to the aggregates.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution.

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.     

Human plasminogen kringle 4. Crystallization and preliminary diffraction data of two different crystal forms

Human plasminogen kringle 4 has been crystallized in two different crystal forms: monoclinic, a = 32.78(3), b = 49.17(2), c = 46.27(3) A, beta = 100.67 degrees, space group P2(1), four molecules/unit cell, two molecules/asymmetric unit; orthorhombic, a = 32.09(7), b = 49.14(6), c = 49.47(9) A, space group P2(1)2(1)2, four molecules/unit cell. Both crystal forms have a large protein fraction (66% for monoclinic and 62% for orthorhombic) and diffract x-rays to 2.0 A resolution. A self-rotation function has been calculated with monoclinic data indicating a non-crystallographic 2-fold rotation approximately parallel to a* (peak height of 14.3 x sigma). Cross-rotation function calculations are in progress utilizing the coordinates of the conserved structure of kringle 1 of prothrombin and plasminogen kringle 4.     

Isolation, characterization, and preliminary X-ray diffraction data for a serine protease from Penicillium cyclopium

The major extracellular protein of Penicillium cyclopium has been isolated from its culture media and purified by ammonium sulfate fractionation, gel, and ion-exchange chromatography. We show this secreted protein to be endopeptidase. The molecular weight is approximately 32,000, the pI is 5.0, and the pH optimum using a variety of protein and synthetic substrates is around 7.0. Inhibition studies show that the protease is not inhibited by pepstatin nor by p-chloromercuribenzoic acid, indicating, respectively, that it is not an aspartyl protease nor a thiol protease. Complete inhibition is observed, however, with phenylmethanesulfonyl fluoride. Three crystal forms suitable for high resolution x-ray diffraction studies have been obtained from this purified protease with reflections being observed to well beyond 3.0 A resolution. One form having a needle morphology is of the orthorhombic crystal class and has space group P2(1)2(1)2(1). The unit cell dimensions are a = 41.9 A, b = 43.2 A, and c = 111.5 A with 1 molecule of the protease occurring in the asymmetric unit. The second form grown at pH values less than 6.0 has a plate morphology, is of orthorhombic space group P2(1)2(1)2(1), and has unit cell dimensions a = 59.12 A, b = 62.33 A, and c = 70.62 A. The third form is polyhedral in habit, is also of space group P2(1)2(1)2(1), and appears when the pH of the mother liquor is greater than 7.0. The cell dimensions of this crystal form are a = 57.07 A, b = 58.82 A, c = 70.79 A, and again there is 1 molecule/asymmetric unit. Three-dimensional structural analysis by x-ray diffraction is now underway. All crystal forms are somewhat denser than the norm having mass to volume ratios of 1.58, 2.00, and 1.85 A3/dalton, respectively.     

D-alanine-D-alanine ligase (ADP) from Salmonella typhimurium. Overproduction, purification, crystallization and preliminary X-ray analysis

The ddlA gene from Salmonella typhimurium coding for D-alanine-D-alanine ligase (ADP-forming) has been subcloned behind the tac promotor in the plasmid pKK223-3, with expression in Escherichia coli JM105. The overexpression system yields 58 mg of active enzyme from 12 g of wet cell paste after 40-fold purification to homogeneity. 5,5'-Dithiobis-(2-nitrobenzoic acid) titrations indicate that all four cysteine residues exist as free thiols. Two crystal forms of the 39,300 Mr enzyme have been produced. A tetragonal form grows at 21 degrees C from 10 to 15% (w/v) polyethylene glycol 8000 in space group P4(1)2(1)2, with two molecules in the asymmetric unit; it has cell constants a = b = 83.8 A, c = 220.0 A, and diffracts to 2.9 A. A monoclinic form grows from 30% (w/v) ammonium sulfate in space group P2(1), with two molecules in the asymmetric unit; it has cell constants a = 60.4 A, b = 102.1 A, c = 64.3 A, beta = 115.7 degrees, and diffracts to 2.2 A resolution.     

Crystal structure of SCO6571 from Streptomyces coelicolor A3(2)

SCO6571 protein from Streptomyces coelicolor A3(2) was overexpressed and purified using Rhodococcus erythropolis as an expressing host. Crystals of selenomethionine-substituted SCO6571 have been obtained by vapor diffusion method. SCO6571 crystals diffract to 2.3 A and were found to belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters a = 84.5, b = 171.6, c = 184.8 A. Six molecules in the asymmetric unit give a crystal volume per protein mass (V(M)) of 2.97 A (3) Da(-1) and solvent content of 58.6 %. The structure was solved by the single wavelength anomalous diffraction (SAD) method. SCO6571 is a TIM-barrel fold protein that assembles into a hexameric molecule with D(3) symmetry.     

The conformation of deamino-oxytocin: X-ray analysis of the 'dry' and 'wet' forms

Two crystal structures of (1 beta-mercaptopropionic acid) deamino-oxytocin are reported. The 'dry form' in space group C2 has cell dimensions a = 27.08 +/- 0.03, b = 9.06 +/- 0.01, c = 22.98 +/- 0.02 A, beta = 102.06 +/- 0.03 with one deamino-oxytocin and six water molecules per asymmetric unit. The 'wet form' in space group P2(1) has cell dimensions a = 27.27 +/- 0.02, b = 9.04 +/- 0.01, c = 23.04 +/- 0.02 A, beta = 102.24 +/- 0.02, with two deamino-oxytocin and 13 water molecules per asymmetric unit. A local twofold parallel to the monoclinic axis gives a pseudo C2 packing. Initial phases of the 'dry form' were calculated by the heavy-atom method from the isomorphous and anomalous difference Pattersons and anomalous difference Fouier synthesis. The structure was refined by using restrained least-squares at 1.2 A resolution to a crystallographic R = 0.10. The molecular replacement method yielded the P2(1) structure that was refined with geometric restraints to R less than 0.09, by using all data to 1.09 A resolution. Deamino-oxytocin consists of a cyclic tocin ring formed by six amino acids, closed by a disulphide bridge, S1-S6, and held by two trans-annular hydrogen bonds N2-O5 and N5-O2 with a type II turn at residues 3 and 4. A flexible tripeptide tail has a loosely hydrogen-bonded type I beta-turn between N9 and O6. The sulphur of cysteine at position 1 is disordered in all the molecules leading to alternative hands of disulphide. The conformational flexibility of Ile 3, Asn 5, Pro 7 side chains and the disulphide bridge is consistent with previous models of oxytocin in which flexibility is necessary for biological activity.     

Crystallization of barley malt alpha-amylases and preliminary x-ray diffraction studies of the high pI isozyme, alpha-amylase 2

alpha-Amylase isozymes 1 and 2 isolated from germinated barley seeds have been crystallized by the hanging- or sitting-drop vapor diffusion technique. Crystals of alpha-amylase 2 suitable for x-ray diffraction analysis were grown at pH 6.7 and 22 degrees C from a solution of 1 mM calcium chloride, 10 mM MES, and 16% saturated ammonium sulfate. The space group is trigonal P3121 (or P3221) with unit cell dimensions a = b = 135.20 A, c = 79.63 A, and probably two molecules per asymmetric unit.     

The tertiary structure of full-length bovine adrenodoxin suggests functional dimers

The three-dimensional X-ray crystal structure of full-length oxidized bovine adrenodoxin (Adx) has been determined at 2.5 A resolution by molecular replacement using a structure of a truncated form as a starting model. Crystals of Adx belong to a primitive monoclinic space group P2(1) with four Adx molecules in an asymmetric unit. The unit cell dimensions are a = 59.44 A, b = 77.03 A, c = 59.68 A, and beta = 94.83 degrees. The structure has been refined to an R factor of 23.5%. Structures of the four molecules of full-length Adx (127 amino acids) in the asymmetric unit were compared with each other and also with that of the truncated Adx (4-108). The overall topology of full-length Adx remains the same as described earlier for the truncated protein. Differences that do occur are almost wholly confined to alternate side-chain conformations that reflect differing lattice contacts made by two proteins. Extensive interactions found between molecules 1 and 2 in the full-length Adx asymmetric unit may reflect the ability of Adx to form dimers in vivo and are consistent with hydrodynamic measurements which show that in solution there is an equilibrium between monomeric and dimeric forms of Adx. Dimerization of Adx could explain why the truncated form has greater affinity for the P450 redox partner than the full-length form. From these results it can be considered that the mechanism of electron transfer is not necessarily the same in different mitochondrial P450 systems.     

Variability in the backbone conformation of cyclic pentapeptides. Crystal structure of cyclic(Gly-L-Pro-D-Phe-Gly-L-Ala)

All the peptide bonds in cyclic(Gly-L-Pro-D-Phe-Gly-L-Ala) are in the trans conformation; however, the peptide bond C'5-N1 is twisted by 19 degrees from planarity (omega 5 = -161 degrees). A Type II beta-turn encompasses the L-Pro-D-Phe residues. Carbonyl oxygens O2, O4 and O5 are directed to the same side of the average plane through the backbone ring and they form hydrogen bonds with N3, N5 and N1, respectively, in adjacent molecules in a stacked column where the adjacent molecules are related by one translational unit. The conformation of the backbone is different from that established in other molecules with the DLDDL chirality sequence. The P21 cell contains two molecules of C21H26N5O5 with a = 4.836(2) A, b = 18.346(8) A, c = 12.464(5) A and beta = 100.05(4) degrees. The R factor for 1382 data with [F0[ greater than 1 sigma is 7.0%.     

Single crystals of chitosan

Lamellar single crystals of chitosan were prepared at 125 degrees C by adding ammonia to a low DP fraction of chitosan dissolved in water. The crystals gave sharp electron diffraction diagrams which could be indexed in an orthorhombic P2(1)2(1)2(1) unit cell with a = 8.07 A, b = 8.44 A, c = 10.34 A. The unit cell contained two anti-parallel chitosan chains and no water molecules. It was found that cellulose microfibrils from Valonia ventricosa could act as nuclei for inducing the crystallization of chitosan on cellulose. This produced a shish-kebab morphology.     

Crystallization of the A-domain of the mannitol transport protein enzyme IImtl.

The A-domain of the mannitol transport protein enzyme IImtl from Escherichia coli (relative molecular mass 16,300) was crystallized, both at room temperature and 4 degrees C, from 40% polyethylene glycol 6000 (pH 8.5 to 9.0) using the hanging-drop method of vapour diffusion. The crystals have the monoclinic space group P2(1), with unit cell dimensions a = 54.0 A, b = 67.0 A, c = 80.9 A and beta = 100.8 degrees. They diffract to 2.6 A resolution. A self-rotation function and self-Patterson suggest that there are four molecules in the asymmetric unit showing mmm symmetry.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Vibrio mimicus.

Single crystals of arylesterase (EC 3.1.1.2) from Vibrio mimicus have been obtained from ammonium sulfate as a precipitant at room temperature for 2 months. The present crystals diffract up to 2.2 A resolution and belong to monoclinic space group P2(1). The cell dimensions are a = 55.65(1) A, b = 53.46(1) A, c = 65.79(1) A, and beta = 106.54(1) degrees. There are two molecules of molecular weight 22 kDa in an asymmetric unit with a solvent content of 43%.