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1.
A bifunctional alpha-amylase/trypsin inhibitor that has two binding sites has been purified from ragi. The inhibitor has been crystallized from its ammonium sulphate solution by the vapour diffusion method. The crystals belong to the orthogonal space group P2(1)2(1)2(1) with unit cell dimensions a = 30.49 A, b = 56.30 A, c = 73.65 A and Z = 4.  相似文献   

2.
Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57.  相似文献   

3.
Crystallization of fragment D from human fibrinogen.   总被引:1,自引:0,他引:1       下载免费PDF全文
Fragment D from human fibrinogen has been crystallized. The fragment, which is composed of three disulfide-linked chains (alpha' beta' gamma' = 88,000), was generated with either plasmin or mild trypsin digestion. The crystals diffracted out to 3.5 A; the space group is P2(1), unit cell dimensions a = 108 A, b = 48 A, c = 167 A, beta = 106 degrees. Fragment D was also co-crystallized with the ligand GPRP-amide, in which case the space group is consistent with P212121, unit cell dimensions a = 476 A, b = 82 A, c = 432 A.  相似文献   

4.
The crystal and molecular structure of trypsin at a transiently stable intermediate step during catalysis has been determined by X-ray diffraction methods. Bovine trypsin cleaved the substrate p-nitrophenyl p-guanidinobenzoate during crystallization under conditions in which the acyl-enzyme intermediate, (guanidinobenzoyl)trypsin, was stable. Orthorhombic crystals formed in space group P2(1)2(1)2(1), with a = 63.74, b = 63.54, and c = 68.93 A. This is a crystal form of bovine trypsin for which a molecular structure has not been reported. Diffraction data were measured with a FAST (Enraf Nonius) diffractometer. The structure was refined to a crystallographic residual of R = 0.16 for data in the resolution range 7.0-2.0 A. The refined model of (guanidinobenzoyl)trypsin provides insight into the structural basis for its slow rate of deacylation, which in solution at 25 degrees C and pH 7.4 exhibits a t1/2 of 12 h. In addition to the rotation of the Ser-195 hydroxyl away from His-157, C beta of Ser-195 moves 0.7 A toward Asp-189 at the bottom of the active site, with respect to the native structure. This allows formation of energetically favorable H bonds and an ion pair between the carboxylate of Asp-189 and the guanidino group of the substrate. This movement is dictated by the rigidity of the aromatic ring in guanidinobenzoate--model-building indicates that this should not occur when arginine, with its more flexible aliphatic backbone, forms the ester bond with Ser-195. As a consequence, highly ordered water molecules in the active site are no longer close enough to the scissile ester bond to serve as potential nucleophiles for hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
Macrophage inflammatory protein 2 (MIP-2) has been crystallized by vapor diffusion of an 11 mg/ml protein solution in 100 mM-ammonium acetate against 30 to 40% polyethylene glycol (average molecular mass of 3350 Da). The crystals belong to space group P2(1)2(1)2(1) and have unit cell dimensions of a = 42.7 A, b = 59.3 A, and c = 100.3 A. The molecular mass of the protein and volume of the unit cell suggest that there are four monomers in the asymmetric unit. A data set to 2.3 A has been collected, and the self-rotation function identifies the presence of a non-crystallographic 2-fold axis.  相似文献   

6.
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.  相似文献   

7.
Crystals of human recombinant factor XIII from the yeast Saccharomyces cerevisiae have been grown from solutions of ammonium sulfate at pH 5.8. The crystals are orthorhombic, with space group P2(1)2(1)2 and unit cell dimensions gamma a = 101.2, b = 182.7, and c = 93.4 A. The asymmetric unit consists of one a2 dimer of molecular mass 166 kDa. A 3.5-A resolution data set for the native protein has been collected. Practical resolution limits for these crystals have not been determined, but reflections have been observed to a Bragg spacing of 2.8-A resolution.  相似文献   

8.
Crystals of the tetrameric xylose isomerase from Streptomyces violaceoniger have been examined by x-ray analysis. Octahedral crystals with a maximum dimension of 0.7 mm were grown from ammonium sulfate solution. They possess the symmetry of P4(1)2(1)2 or P4(3)2(1)2 space groups, which are crystallographically indistinguishable. The unit cell dimensions are a = b = 140 A and c = 134 A. There is one tetramer of molecular weight 160,000 per asymmetric unit. The crystals diffract to 2.2 A.  相似文献   

9.
A toxic lectin, ricin D, present in the seeds of Ricinus communis has been purified and crystallized in a form suitable for high resolution crystallographic structure studies. This protein is different from a previously found form of ricin (also present in the same seeds), the only ricin for which a preliminary x-ray investigation has been reported so far. Ricin D crystallizes from an aqueous solution in an orthorhombic unit cell of symmetry P2(1)2(1)2(1) and a = 79.0, b = 114.7, and c = 72.8 A. The asymmetric unit contains one molecule with an average molecular weight of 62,400. The crystal is fairly stable to x-radiation and has a water content of approximately 54% by volume. It appears to comprise two closely related species of proteins, the major species corresponding to recin D and the other presumably corresponding to a deamidation product of ricin D. The two species have nearly identical molecular size and amino acid compositions, but different charges.  相似文献   

10.
Malate dehydrogenase from mutant strain F428 of the thermophilic bacterium Thermus flavus has now been crystallized from polyethylene glycol 8000 in a form suitable for diffraction studies. The protein crystallizes in the orthorhombic P2(1)2(1)2(1) space group with unit cell dimensions a = 71.8 A, b = 88.6 A, c = 119.0 A. The asymmetric unit consists of one homodimer of molecular mass 67,000 Da. The X-ray diffraction extends beyond 1.7 A and a full data set to 1.9 A has been collected.  相似文献   

11.
Crystals of methylamine-treated alpha 2-macroglobulin (alpha 2M-MA), alpha 2-macroglobulin in complex with two molecules of trypsin, alpha 2M-T2, one molecule of plasmin, alpha 2M-PL, and one molecule of plasmin followed by methylamine-treatment, alpha 2M-PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of alpha 2M-MA diffracted to at least 9 A resolution, crystals of alpha 2M-T2 diffracted to 10 A resolution and crystals of alpha 2M-PL and alpha 2M-PL(MA) diffracted to 11 A resolution. For alpha 2M-MA the cell parameters were determined as: a=b=257 A, c=555 A; and for alpha 2M-T2 as: a=b=247 A, c=559 A. For both preparations the space group was I4(1)22. As estimated from density measurements, the crystals of alpha 2M-MA and alpha 2M-T2 contain one 360 kDa alpha 2M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight, Vm, was estimated at 5.6 A3/Da. The crystal parameters of alpha 2M-PL and alpha 2M-PL(MA) were not determined.  相似文献   

12.
Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.  相似文献   

13.
Crystals of a complex formed between the alpha/beta-tubulin heterodimer and SCG10, a neuron-specific growth-associated protein, have been obtained by the hanging drop method. They belong to the space group P2(1)2(1)2(1), with unit cell parameters a = 56 A, b = 353 A, c = 466 A and four molecular complexes in the asymmetric unit. A complete X-ray diffraction data set to 6.1 A resolution has been collected using synchrotron radiation. This represents a challenging opportunity to study at a molecular level the structure-function relationships between a microtubule-destabilizing protein, SCG10, and tubulin.  相似文献   

14.
Crystals of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar) have been grown from ammonium sulphate solution at pH 5.0. Two crystal forms suitable for X-ray structure analysis, obtained from a hanging-drop experiment, have been characterized. Both belong to space-group P22(1)2(1) with cell dimensions a = 39.2 A, b = 62.4 A, c = 84.6 A and a = 31.4 A, b = 74.8 A, c = 83.5 A, for forms I and II, respectively. Intensity data to 1.82 A have been collected for crystal form I on a CAD4 diffractometer, and initial phases have been obtained by molecular replacement methods. The conventional R-factor after two rounds of model building and subsequent refinement is 0.25 for data between 6.0 and 2.0 A. So far no water molecules have been included in the model.  相似文献   

15.
Methane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of methane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition alpha 2 beta 2 gamma 2. The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol, lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group P2(1)2(1)2(1), with one dimer of relative molecular mass M(r) = 252,000 in the asymmetric unit. The unit cell dimensions are a = 62.6 A, b = 110.1 A, c = 333.5 A. The crystals diffract uniformly beyond 2.5 A resolution. Crystals of the related hydroxylase from Methylosinus trichosporium OB3b have also been obtained.  相似文献   

16.
Crystallization of recombinant rat cathepsin B   总被引:1,自引:0,他引:1  
A glycosylation-minus mutant of rat cathepsin B expressed in yeast has been purified and crystallized. X-ray diffraction data have been collected and molecular replacement for solving the structure is in progress. The space group for the recombinant rat cathepsin B was determined to be P2(1) with unit cell dimensions alpha = 62.2 A, b = 90.19 A, c = 47.07 A, and beta = 97.43 degrees. A unit cell contains 4 molecules and 2 molecules per asymmetric unit.  相似文献   

17.
An insect-specific toxin from the venom of the scorpion Androctonus australis Hector has been crystallized. The crystals are orthorhombic, space groups P2(1)2(1)2(1), with cell dimensions a = 66.4 A, b = 52.5 A and c = 36.1 A. Calculations based on the unit cell volume and toxin molecular mass suggest that there are two molecules in the asymmetric unit.  相似文献   

18.
TE33 is an Fab fragment of a monoclonal antibody raised against a 15-residue long peptide (CTP3), corresponding in sequence to residues 50-64 of the cholera toxin B subunit. Crystals of the complex between TE33 and CTP3 have been grown from 20% (w/v) polyethylene glycol-8000 at pH 4.0. The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 104.15, b = 110.61, and c = 40.68 A. X-Ray data have been collected to a resolution of 2.3 A. The asymmetric unit contains one molecule of Fab and one molecule of CTP3. The presence of CTP3 has been demonstrated by fluorescence quenching of the dissolved crystal after X-ray data collection. A molecular replacement solution was found based on the coordinates of DB3, an antiprogesterone Fab fragment.  相似文献   

19.
Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3 M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.  相似文献   

20.
Single crystals of hydrogenase from Desulfovibrio vulgaris Miyazaki F   总被引:2,自引:0,他引:2  
The hydrogenase solubilized from the particulate fraction from Desulfovibrio vulgaris Miyazaki F (IAM 12604) has been crystallized. Although the solubilized hydrogenase purified by the previous method (Yagi, T., Kimura, K., Daidoji, H., Sakai, F., Tamura, S., and Inokuchi, H. (1976) J. Biochem. (Tokyo) 79,661-671) revealed a single band upon disc electrophoresis, it could not be crystallized. The apparently homogeneous hydrogenase has been separated into three components of similar molecular weights by high performance liquid chromatography on DEAE-Toyopearl. Each hydrogenase component was successfully crystallized by means of the vapor diffusion method with polyethylene glycol or 2-methyl-2,4-pentanediol as a precipitating agent. Seeding procedure is necessary to grow an x-ray grade crystal. Preliminary x-ray experiments reveal that crystals grown from one component are in space group of P2(1)2(1)2(1) with a = 102.1(1), b = 126.8 (3), and c = 66.9(1) A. The unit cell volume of 8.66 X 10(5) A3 suggests that it contains one molecule/asymmetric unit (Vm = 2.43). The crystals grown from another component are in the same space group with a = 99.6(1), b = 126.8(3), c = 66.9(1) A, and the unit cell volume is 8.45 X 10(5) A3 (Vm = 2.37). The crystals diffract more than 2.5 A and are suitable for complete crystal analysis. Up to 4 A resolution native data have been collected on a diffractometer.  相似文献   

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