Characterization of two crystal forms of human defensin neutrophil cationic peptide 1, a naturally occurring antimicrobial peptide of leukocytes

Two orthorhombic crystal forms (P2(1)2(1)2, a = 30.5 A, b = 44.5 A, c = 40.5 A; I2(1)2(1)2(1) (or I222), a = 30.1 A, b = 66.5 A, c = 35.5 A) of human neutrophil cationic peptide 1 have been characterized. The P2(1)2(1)2 form contains two peptides (Mr = 3425) per asymmetric unit; the I2(1)2(1)2(1) form contains one peptide per asymmetric unit. Both crystal forms diffract to beyond 1.8 A resolution.     

Crystallization and characterization of Chromatium vinosum cytochrome c'

The dimeric high spin c-type cytochrome c' from Chromatium vinosum has been crystallized and the crystals characterized by x-ray diffraction. This cytochrome c' exhibits ligand-controlled dissociation from a dimer to a monomer upon binding carbon monoxide and represents an opportunity to obtain unique information concerning cooperativity in heme proteins. The C. vinosum cytochrome c' protein crystals are grown from polyethylene glycol 4000 and grow in both space group P2(1)2(1)2(1) (a = 49.2, b = 56.7, c = 98.8 A) and space group P2(1) (a = 55, b = 94, c = 50, beta = 106.1 A) depending upon the growth rate, with the P2(1)2(1)2(1) form favored at slower growth rates. The high resolution (2.0 A) atomic structure of the P2(1)2(1)2(1) form is being determined.     

Proteins of the Bacillus stearothermophilus ribosome. Crystallization of proteins L30 and S5

Proteins L30 and S5 from the 50 S and 30 S subunits, respectively, of the Bacillus stearothermophilus ribosome have been crystallized. L30 crystals are tetragonal and the space group is P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 46.3 A and c = 61.4 A. S5 crystals are trigonal with the space group P3(1)21 (or P3(2)21) and cell dimensions a = b = 59.3 A and c = 109.8 A. In both cases, there appears to be a single molecule in the asymmetric unit.     

Stabilization of P450 2B4 by its association with P450 1A2 revealed by high-pressure spectroscopy

We studied the effect of intermolecular interactions between cytochromes P450 1A2 (CYP1A2) and 2B4 (CYP2B4) on the barotropic inactivation of the ferrous carbonyl complexes of the hemoproteins. When taken separately, these hemoproteins reveal quite distinct barotropic behavior. While the 2B4(Fe(2+))-CO complex is very sensitive to hydrostatic pressures and undergoes P450 --> P420 transition at rather low pressures (P(1/2) = 297 MPa, DeltaV(0) = -61 ml/mol), the 1A2(Fe(2+))-CO is extremely resistant to barotropic inactivation. Only about 8% of the 1A2 was exposed to pressure-induced P450 --> P420 transition (P(1/2) = 420 MPa, DeltaV(0) = -28 ml/mol). The formation of the mixed oligomers of 2B4 and 1A2 was found to have a dramatic effect on the barotropic behavior of 2B4. In the heterooligomers of 1A2 and 2B4, the 2B4 hemoprotein appears to be largely protected from barotropic inactivation. In 1:1 mixed oligomers no more than 25% of the total P450 content undergoes P450 --> P420 inactivation with the molar reaction volume value (DeltaV(0) = -26 ml/mol) similar to those found for pure 1A2. Moreover, interactions between 1A2 and 2B4 results in a displacement of the Soret band of the ferrous carbonyl complex of CYP2B4 to shorter wavelength (from 451.3 to 448.4 nm) and largely strengthens the dependence of the Soret band wavenumber on hydrostatic pressure below 200 MPa. This effect suggests an important hydration of the CYP2B4 heme moiety in response to the interactions with CYP1A2. We discuss these results in terms of the hypothesis that the heterooligomerization of cytochromes P450 in microsomes plays an important role in the control of the activity and coupling of the microsomal monooxygenase.     

Crystallization of and preliminary X-ray data for the mouse major urinary protein and rat alpha-2u globulin.

Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A, c = 62.7 A, beta = 95.1 degrees) with four subunits/asymmetric unit, while MUP gave crystals in P4(1)2(1)2 or P4(3)2(1)2 (a = 57.3 A, c = 109.9 A) with one subunit/asymmetric unit. Both crystal forms diffract beyond 2.8 A resolution.     

Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB.

The major cold shock protein from Bacillus subtilis (CspB) was overexpressed using the bacteriophage T7 RNA polymerase/promoter system and purified to apparent homogeneity from recombinant Escherichia coli cells. CspB was crystallized in two different forms using vapor diffusion methods. The first crystal form obtained with ammonium sulfate as precipitant belongs to the trigonal crystal system, space group P3(1)21 (P3(2)21) with unit cell dimensions a = b = 59.1 A and c = 46.4 A. The second crystal form is tetragonal, space group P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 56.9 A and c = 53.0 A. These crystals grow with polyethylene glycol 4000 as precipitant.     

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.     

Relationships between TGFbeta proteins and oxygen concentrations inside the first trimester human gestational sac

In early pregnancy, the O(2) gradient between the maternal circulation and the gestational sac tissues modulates trophoblast biological functions. The aim was to evaluate if placental partial pressure of oxygen (PaO(2)) modulates in vivo synthesis of specific placental proteins inside the first trimester gestational sac. Matched samples of peripheral venous blood, blood from the placental bed (PB), coelomic fluid (CF) and placental tissue were obtained in 37 normal pregnancies at 6-12 weeks gestation. PaO(2) was measured in PB and CF using an IRMA blood gas monitor. Inhibin A, activin A, sEng, PlGF, sFlt-1 and free VEGF concentrations were measured in all samples. HSP 70 was measured in placental extracts. ANOVA showed approximately 60% increase in PB PaO(2) (P = 0.02) between after 10 weeks gestation. Unpaired Student's T-test between two groups (6-9 weeks vs 9-12 weeks) shows a significant increase in MS Activin A (P = 0.001), CF activin A (P<0.001), MS P1GF (P = 0.001), CF PlGF (P<0.001), MS sFLT-1 (P = 0.03), CF sFLT-1 (P = 0.01), HSP 70 in placental extracts (P = 0.04) and a significant decrease in PB inhibin A levels (P<0.001) and PB sFLT-1 (P = 0.02) . Multiple correlation analysis showed a significant negative correlation between PB inhibin A levels and gestation (r = -0.45, P<0.05) and PB PaO(2) (r = -0.5, P = 0.008) and also between sFLT-1 and PB PaO(2) (P = 0.03). There was a positive correlation (P<0.01) between PlGF, sEng and VEGF levels in the placental extracts. Our results indicate a direct relationship in the early intrauterine PaO(2) in vivo and inhibin A and sFLT-1 concentrations confirming our hypothesis that specific placental proteins are regulated by intrauterine O(2) tension.     

Crystallization and characterization of the high molecular weight form of nerve growth factor (7 S NGF)

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.     

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 diffraction analysis of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the bacterium Cellulomonas fimi.

Single crystals of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the cellulolytic bacterium Cellulomonas fimi, have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique. The crystals, which diffract to better than 2.0 A resolution, belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have cell constants: a = b = 88.21 A, c = 81.10 A; alpha = beta = gamma = 90 degrees.     

Crystallization of a Fe,Zn superoxide dismutase from the archaebacterium Thermoplasma acidophilium

The novel Fe,Zn superoxide dismutase from the archaebacterium Thermoplasma acidophilium has been crystallized in space groups P1, P2(1) and P2(1)2(1)2, with 2,4 and 1/2 of an 84,000 Mr tetramer, respectively, estimated to be in the asymmetric unit of the unit cell. The orthorhombic crystals, which have unit cell dimensions a = 84.2 A, b = 72.7 A, c = 67.8 A, diffract X-rays to at least 2.0 A and are suitable for a determination of the three-dimensional structure of the Fe,Zn superoxide dismutase.     

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.     

Enantiomerically pure quaternary ammonium salts with a chiral alkyl chain N(CH3)(n-C3H7)2(sec-C4H9)I: synthesis and physical studies

A pair of enantiomerically pure quaternary ammonium salts with a chiral side chain, methyl-(R)-(1-methylpropyl)di(n-propyl)ammonium iodide 1 and methyl-(S)-(1-methylpropyl)di(n-propyl)ammonium iodide 2, and the related racemate, methyl-(rac)-(1-methylpropyl)di(n-propyl)ammonium iodide 3, were synthesized through a reductive alkylation procedure, starting from enantiomerically pure and, also, racemic forms of (rac)-(1-methylpropyl)amine. A spectroscopic chiroptical signature in solution was provided by the Raman optical activity spectra of compounds 1 and 2. The crystallographic structures of 1, 2, and 3 were examined by single crystal X-ray diffraction. 1 crystallizes in the tetragonal space group P4(3)2(1)2 (no. 96), a = b = 12.826 (2) A, c = 17.730 (2) A, V = 2916.9 (5) A(3), Z = 8, Flack coefficient 0.04 (2). 2 crystallizes in the tetragonal space group P4(1)2(1)2 (no. 92), a = b = 12.842 (1) A, c = 17.749 (2) A, V = 2927.0 (5) A(3), Z = 8, Flack coefficient 0.05 (2). The crystal structures and space groups for 1 and 2 are enantiomorphs and the crystallographic investigation confirmed the absolute configuration of the stereocenter in both compounds. 3 crystallizes in the monoclinic space group P2(1)/n(no. 14), a = 8.178 (1) A, b = 14.309 (2) A, c = 12.328 (2) A, beta = 96.811 (6) degrees, V = 1432.4 (2) A(3), Z = 4.     

Crystallization and preliminary X-ray analysis of a fungal endoglucanase I.

An endoglucanase I (EG1) from a fungal source (Humicola insolens) has been crystallized in a number of forms suitable for X-ray diffraction analysis. Four crystal forms have been grown from various precipitants using vapour phase diffusion methods in hanging drops. Three of these crystal forms diffract to beyond 2.5 A resolution. Two forms, obtained from ammonium sulphate at pH 5.4, or 8.0, grow as tetragonal bipyramids in space group P4(1)22 or P4(3)22, with approximate cell dimensions a = b = 102 A, c = 282 A. The other crystal forms were grown from polyethylene glycol 8000 at pH 8.0. One grows as monoclinic plates, space group P2(1), with cell dimensions a = 66.9 A, b = 75.2 A, c = 86.9 A and beta = 102.9 degrees and the other as long hexagonal rods in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 119 A, c = 83 A.     

Preliminary crystallographic studies of two C-terminally truncated copper-containing nitrite reductases from Achromobacter cycloclastes: changed crystallizing behaviors caused by residue deletion

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.     

Tetragonal crystals of canine myeloperoxidase suitable for X-ray structural analysis

A new tetragonal crystal form of canine myeloperoxidase grown by precipitation with ammonium sulfate is described. The space group is P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions. a = b = 133.0 A, c = 203.6 A, and a single molecule in the asymmetric unit. The crystals diffract to Bragg spacings of 2.5 A and are suitable for a medium-resolution structure determination.     

Crystallization and preliminary X-ray studies of human recombinant interleukin-2

Two different forms of crystals (potentially) suitable for x-ray structure analysis were obtained for recombinant human interleukin-2 (IL-2) using ammonium sulfate as a precipitant in the pH range of 6.3-7.3 (in the case of hexagonal bipyramidal crystals) and 4.5-5.5 (in the case of plate crystals). The hexagonal bipyramidal crystal belongs to a hexagonal space group P6(2)22 or P6(4)22 with a = b = 105.8 A and c = 122.2 A. The crystal diffracts up to 3.4 A resolution and contains 2 or 3 IL-2 molecules in an asymmetric unit. The plate crystal belongs to an orthorhombic space group P2(1)2(1)2 with a = 47.9 A, b = 79.6 A, and c = 31.9 A. The crystal diffracts up to 2.5 A resolution and contains only 1 IL-2 molecule in an asymmetric unit. These facts reconfirmed crystallographically the high homogeneity of the present preparation of human recombinant IL-2.     

Crystallization of a photosensitive nitrile hydratase from Rhodococcus sp. N-771.

A photosensitive nitrile hydratase from Rhodococcus sp. N-771 has been crystallized in two different crystal forms in its inactive form. One crystal form belongs to an orthorhombic space group P2(1)2(1)2 with unit cell dimensions of a = 117.4 A, b = 145.7 A and c = 52.1 A, and the other form belongs to a hexagonal space group P6(3)22 with unit cell dimensions of a = 110.2 A and c = 412.1 A.     

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.