Structural Study of Escherichia coli NAD Synthetase: Overexpression, Purification, Crystallization, and Preliminary Crystallographic Analysis

Escherichia coli NAD synthetase was overexpressed and purified to homogeneity. The recombinant protein was active in an in vitro enzyme assay. The enzyme required approximately 1.5 mM magnesium for optimal activity. The pH optimum was found to be 8.0-8.5. The recombinant protein was crystallized at room temperature using the hanging-drop vapor diffusion technique with 1.5 M lithium sulfate, 0. 1 M Hepes buffer at pH 7.5 as precipitant. The protein was also crystallized in the presence of its substrates, nicotinic acid adenine dinucleotide and adenosine triphosphate under similar conditions. These crystals diffract to 2.0-A resolution and belong to trigonal space group P3(1)21 with unit cell dimensions of a = b = 91.766, c = 74.17 A and alpha = beta = 90 degrees, gamma = 120 degrees. The structure of the complex has been determined using the molecular replacement method.     

Crystallization and preliminary x-ray diffraction analysis of three mastoparans

Mastoparans are tetradecapeptides found to be the major component of wasp venoms. These peptides possess a variety of biological activities. Three related mastoparans, mastoparan from Polistes jadwagae (MP-PJ), mastoparanX (MP-X) and its carboxyl-free C-terminal form (MP-X-COO-), were crystallized. X-ray diffraction data for them were collected at resolutions of 1.2 A, 2.0 A and 3.3 A respectively.     

Crystallization and preliminary X-ray diffraction studies of a deglycosylated glucose oxidase from Aspergillus niger

Deglycosylation was shown to be an important prerequisite step for the crystallization of glucose oxidase from Aspergillus niger. Whereas the glycosylated enzyme could not be crystallized, crystals of the deglycosylated enzyme suitable for X-ray diffraction analysis were reproducibly grown in the presence of 1.6 M-ammonium sulphate and octanetriol at pH 5.3 to 5.6. The crystals belong to the space group P3(1)21 or P3(2)21 with refined lattice constants of a = 66.5 A and c = 214.4 A, indicating a cell content of one monomer per asymmetric unit of the crystal. Crystals diffract to at least 2.5 A resolution. Cleavage of 95% of its carbohydrate moiety affected the kinetics of glucose oxidation, stability at low pH and some electrophoretic properties of glucose oxidase, such as molecular mass and the number of isoelectric forms. However, other properties, such as thermal stability, pH and temperature optima of activity were not affected.     

Pertussis toxin-insensitive effects of mastoparan,a wasp venom peptide,in PC 12 cells

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, modifies the secretion of neurotransmitters and hormones from a variety of cell types. Mastoparan interacts with heterotrimeric guanine nucleotide-binding proteins (G proteins) such as G_i and G_o, which are ADP-ribosylated by pertussis toxin (PTX) and thereby uncoupled from receptors. Previously, some of the effects of mastoparan including secretion were reported to be modified selectively by PTX but not by cholera toxin (CTX). In the present study, we examined the influence of bacterial toxins on the effects of mastoparan in PC12 cells. Mastoparan stimulated [³H]noradrenaline (NA) release from prelabeled PC12 cells in the absence of CaCl₂, although high K⁺ or ATP stimulated the release in a Ca²⁺-dependent manner. Pretreatment with CTX, not PTX, for 24 h inhibited mastoparan-stimulated [³H]NA release. Mastoparan inhibited forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner, although mastoparan had no effect by itself. Pretreatment with PTX completely abolished the inhibitory effect of carbachol via G_i on cyclic AMP accumulation and partially reduced the effect of mastoparan. However, the inhibitory effect of 20 μM mastoparan was not modified by pretreatment with PTX. Thus, we investigated the effect of mastoparan on CTX-catalyzed [³²P]ADP-ribosylation of proteins in PC12 cells. A subunit of CTX (CTX-A) catalyzed [³²P]ADP-ribosylation of many proteins in the cytosolic fraction of PC12 cells. One of these was a 20 kDa protein, named ADP-ribosylating factor (ARF). The addition of mastoparan to assay mixtures inhibited ADP-ribosylation of many proteins including ARF and CTX-A in the presence of the cytosolic fraction. In the absence of the cytosolic fraction, however, mastoparan slightly enhanced ADP-ribosylation of bovine serum albumin and auto-ADP-ribosylation by CTX-A. Mastoparan did not inhibit ADP-ribosylation of the α subunit of G_s in the membrane fraction. These findings suggest that (1) mastoparan interacts with PTX-insensitive and CTX-sensitive factor(s) to stimulate NA release, and (2) mastoparan interacts with ARF inhibiting its activity to enhance the ADP-ribosylation reaction by CTX. ARF may be an exocytosis-linked G protein. © 1996 Wiley-Liss, Inc.     

Preparation of human manganese superoxide dismutase by tri-phase partitioning and preliminary crystallographic data

Human liver manganese superoxide dismutase has been purified by a short procedure that includes a tri-phase partitioning step to provide materials that can be crystallized from ammonium sulfate. X-ray diffraction studies at 3 A resolution show that the crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with cell dimensions a = b = 81.1 A, c = 242.2 A. Manganese superoxide dismutase levels as determined by enzymatic assay as well as by enzyme-linked immunosorbent assay indicated that considerable variations occur in different livers but the total superoxide dismutase activity (Mn superoxide dismutase plus Cu,Zn superoxide dismutase) seems to be kept at constant values.     

Crystallization, activity assay and preliminary X-ray diffraction analysis of the uncleaved form of the serpin antichymotrypsin.

Crystals of recombinant wild-type antichymotrypsin have been prepared by the method of vapor diffusion with polyethylene glycol 4000 as a precipitant at pH 5.7. Two crystal forms are observed. One form belongs to tetragonal space group P4(3)2(1)2 (or P4(1)2(1)2) and has unit cell dimensions a = b = 126 A, c = 243 A, with two molecules in the asymmetric unit. The other crystal form belongs to orthorhombic space group P2(1)2(1)2(1) and has unit cell parameters of a = 73 A, b = 78 A and c = 80 A, with one molecular in the asymmetric unit. Diffraction intensity measurements have been made on the tetragonal crystal form to a limiting resolution of 4.1 A, and reflections have been observed on X-ray still photographs to a limiting resolution of 2.5 A for the orthorhombic form. An activity assay of redissolved tetragonal form crystals indicates that the uncleaved, functional serpin has been crystallized.     

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system. Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 A. The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.     

Crystallization of insecticyanin from the hemolymph of the tobacco hornworm Manduca sexta L. in a form suitable for a high resolution structure determination

Insecticyanin, a blue biliprotein from the tobacco hornworm Manduca sexta, has been crystallized in a form suitable for a high resolution x-ray analysis. The crystals grow by vapor diffusion against solutions of polyethylene glycol 8000 at pH 5.5. They belong to the space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 115.0 A; c = 71.1 A. Insecticyanin is believed to be a tetramer in solution; there are two subunits per asymmetric unit. The crystals diffract to at least 2.2 A resolution and appear reasonably resistant to radiation damage.     

Crystallization and preliminary X-ray diffraction studies of the engrailed homeodomain and of an engrailed homeodomain/DNA complex

The homeodomain from the engrailed protein of Drosophila has been crystallized from ammonium phosphate at pH 6.8. The crystals form in space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 44.8 A and c = 118.2 A. These crystals diffract to 1.8 A resolution. A complex containing the engrailed homeodomain and a duplex DNA site also has been crystallized. The cocrystals form in space group C2 with a = 131.2 A, b = 45.5 A, c = 72.9 A and beta = 119.0 degrees. These crystals diffract to 2.6 A resolution.     

Preliminary X-ray crystallographic study of malate dehydrogenases from the thermoacidophilic Archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius

Malate dehydrogenases from the thermoacidophilic Archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius have been crystallized and characterized by X-ray diffraction measurements. Crystals of the enzyme from T. acidophilum display space-group symmetry P2(1), a = 63 A, b = 135 A, c = 83 A and beta = 105 degrees; they scattered to approximately 4 A resolution. Two crystal modifications of malate dehydrogenase from S. acidocaldarius were characterized; one displayed trigonal symmetry corresponding to space groups P321, P3(1)21 or P3(2)21 with lattice parameters a = 151 A and c = 248 A and with resolution approximately to 5 A, whereas the other modification displayed space group symmetry I23 or I2(1)3 with lattice parameters a = 129 A and approximately 4.5 A resolution.     

Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli.

Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A.     

Crystallization and the crystal properties of the oxygen-evolving photosystem II from Synechococcus vulcanus

A photosystem II (PSII) complex highly active in oxygen evolution was purified and crystallized from a thermophilic cyanobacterium, Synechococcus vulcanus. The PSII complex in the crystals contained the D1/D2 reaction center subunits, CP47 and CP43 (two chlorophyll-binding core antenna proteins of photosystem II), cytochrome b-559 alpha- and beta-subunits, several low molecular weight subunits, and three extrinsic proteins, that is, 33 and 12 kDa proteins and cytochrome c-550. The PSII complex also retained a high rate of oxygen evolution. The apparent molecular mass of the PSII in the crystals was determined to be 580 kDa by gel filtration chromatography, indicating that the PSII crystallized is a dimer. The crystals diffracted to a maximum resolution of 3.5 A at a cryogenic temperature using X-rays from a synchrotron radiation source, SPring-8. The crystals belonged to an orthorhombic system, and the space group was P2(1)2(1)2(1) with unit cell dimensions of a = 129.7 A, b = 226.5 A, and c = 307.8 A. Each asymmetric unit contained one PSII dimer, which gave rise to a specific volume (V(M)) of 3.6 A(3)/Da based on the calculated molecular mass of 310 kDa for a PSII monomer and an estimated solvent content of 66%. Multiple data sets of native crystals have been collected and processed to 4.0 A, indicating that our crystals are suitable for structure analysis at this resolution.     

Crystallization and preliminary analysis of telokin, the C-terminal domain of myosin light chain kinase

Telokin, an acidic protein related to the C-terminal portion of smooth muscle myosin light chain kinase from turkey gizzard has been crystallized in a form suitable for a high-resolution diffraction analysis. The crystals were grown from solutions of polyethylene glycol 8000 using the hanging-drop vapor diffusion method. They belong to the trigonal space group P3(1)21 or P3(2)21 with cell parameters a = 64.0 A, c = 59.4 A and diffract to at least 2.7 A resolution.     

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 studies on Sulfolobus acidocaldarius ferredoxin.

Ferredoxin from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius, has been crystallized. The space group is P4(3)2(1)2 or P4(1)2(1)2 and the cell dimensions are a = b = 50.12 A and c = 69.52 A. The Vm value is calculated to be 1.88 A3/Da, assuming one molecule per asymmetric unit. The crystal diffracts X-rays beyond 2.0 A resolution.     

Crystallization and preliminary X-ray analysis of a quadruple mutant of staphylococcal nuclease

A quadruple mutant of staphylococcal nuclease, nuclease (V66L/G79S/G88V/L108V), has been crystallized in a form well suited to moderate-to-high resolution x-ray diffraction analysis. This mutant is highly unstable; only about 20% of the protein in solution at room temperature is in its folded form. Under the crystallization conditions, the protein exhibits circular dichroism properties similar to, but not identical with, those of native wild type protein. The crystals belong to the space group P6(1)22 or P6(5)22 with unit cell dimensions of a = b = 61.1 A, c = 170.1 A and diffract to at least 2.5 A resolution. A data set complete to 3.7 A resolution has been collected and processed; attempts to determine the structure using molecular replacement techniques are under way.     

Facilitation of phospholipase A2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom

The effect of mastoparan, Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-LeuNH2, and related peptides on the release of arachidonic acid from egg yolk lecithin liposomes, rat peritoneal mast cells, and cultured human fibroblasts was studied. In unsonicated liposomes, labeled with 1-stearoyl-2[1-14C]arachidonyl-sn-glycero-3-phosphocholine, 5 X 10(-5) M mastoparan caused a 12-, 15-, and 50-fold increase in the production of arachidonic acid catalyzed by phospholipase A2 from bee venom, eastern diamondback rattlesnake and porcine pancreas, respectively. The stimulant effect of mastoparan and related peptides was dose-dependent and further enhanced by sonication of liposomes. In contrast, melittin, while stimulating the production of arachidonic acid by phospholipase from bee venom, was inactive with the rattlesnake and pancreatic enzymes. Melittin was also only weakly active with liposomes containing stearic acid in place of arachidonic acid. Like melittin, mastoparans stimulated phospholipase activity in tissue homogenates and caused a dose-dependent release of arachidonic acid from rat peritoneal mast cells and cultured human fibroblasts prelabeled with [14C]arachidonic acid. The heptapeptide fragments mastoparan 1-7 and mastoparan 8-14, and succinylated mastoparan were ineffective. The results suggest that mastoparan and related peptides in insect venoms act, at least in part, by stimulating phospholipase activity.     

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.     

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.     

Crystal structure of mastoparan from Polistes jadwagae at 1.2 A resolution

Mastoparans, a group of amphiphilic tetradecapeptides, are the major peptides in social wasp venoms and possess a variety of biological activities. Here we report the first crystal structure of mastoparan from Polistes jadwagae (MP-PJ) at 1.2 A resolution. The crystals belong to the space group P2(1) with eight molecules in an asymmetric unit. In contrast to the previous observations that the alpha-helical conformation only exists in the membrane-bound state of mastoparans, all of the MP-PJ molecules are in possession of the alpha-helical conformation even in the absence of trifluorethanol or detergents in the crystallization system. The high-resolution structure enables us to compare the conformation differences of MP-PJ with NMR results of other mastoparans. Together with biochemical results, we propose that the interactions between mastoparan molecules play an important role in forming the alpha-helical conformation, which is highly related to their biological activities.