X-Ray Analysis of New Crystal Forms of the Sweet Protein Thaumatin

Abstract

Thaumatin is a plant protein that in the mature form contains 8 disulfide bonds and 207 amino acids. Several forms of this protein occur naturally and each elicits an intense sweetness sensation when tasted in microgram quantities. The two major forms of thaumatin are easily separable by ion exchange chromatography. Crystals of the two proteins (designated here A and B) have been grown by vapor equilibration from solutions containing polyethylene glycol and examined by X-ray diffraction. The thaumatin A crystals are of space group P2₁2₁2₁ with a=44.3Å, b=63.7Åand c=72.7A. The crystals of thaumatin B are of space group C2 with a = 117.7Å, b=44.9Å, and c=38.0Å and β=94.0°. Both crystals diffract to well beyond 2.3Å and appear suitable for high resolution structure analysis. Four heavy atom derivatives of thaumatin B have been generated and diffraction data to 4Å resolution have been collected. This work is designed to provide a basis for studying the 3-dimensional structure of more than 100 genetically generated thaumatin derivatives, several of which show enhanced stability and improved taste characteristics.     

Preliminary X-ray diffraction studies of the putative catalytic domain of gamma delta resolvase from Escherichia coli

Two crystal forms of the putative catalytic domain (residues 1-140) of gamma delta resolvase from Escherichia coli have been obtained. Type I is isomorphous with crystals of the intact protein, and type II is suitable for high resolution structure analysis. Type II crystals belong to the orthorhombic space group C222(1), with a = 76.8 A, b = 191.3 A, and c = 63.4 A. They contain two molecules (15,500 daltons each)/asymmetric unit and show diffraction beyond 2.7-A resolution. Calculation of a rotation function using 7-A data shows the orientation of the noncrystallographic axes.     

Crystallization and preliminary diffraction data for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii

Pink crystals of methylmalonyl-CoA mutase from Propionibacterium shermanii, a coenzyme B12 (5'-deoxyadenosylcobalamin)-dependent enzyme, have been obtained by the hanging-drop method in two different forms. One form lies in the space group P21, with unit cell dimensions a = 122 A, b = 160 A and c = 90 A, with beta = 104 degrees (1 A = 0.1 nm). There are two alpha beta dimers in the asymmetric unit. The crystals diffract to 3.2 A resolution and are suitable for high resolution X-ray diffraction studies.     

Preliminary X-ray crystallography studies of recombinant human interleukin-1 alpha. Purification and structural characterization

Human interleukin-1 alpha, cloned and expressed in E. coli, has been purified and structurally characterized by various physiochemical methods, including mass spectrometry. The recombinant protein has been crystallized by the hanging drop vapor diffusion method using dimethyl sulfoxide as the precipitating agent. The space group is P2(1)2(1)2(1). Unit cell dimensions are a = 44.1, b = 57.1, and c = 61.7 A and alpha = beta = gamma = 90 degrees. The crystals diffract to beyond 1.7 A and are suitable for high resolution data collection. Native diffraction data were collected. Screens for heavy atom derivatives have been initiated.     

Preliminary crystallographic studies on human apo-lactoferrin in its native and deglycosylated forms

Human apo-lactoferrin in both native and deglycosylated forms has been purified, and crystals obtained by dialysis against low ionic strength buffer solutions. The crystals of native apo-lactoferrin are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 222.0 A, b = 115.6 A, c = 77.8 A and have two protein molecules per asymmetric unit. Two crystal forms of deglycosylated apo-lactoferrin have been obtained. One is orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 152.1 A, b = 94.6 A, c = 55.8 A. The second is tetragonal, space group I4, with cell dimensions a = b = 189.4 A, c = 55.1 A. Both of the latter have only one molecule per asymmetric unit, and are suitable for high-resolution X-ray structure analysis.     

Crystallization of P2 myelin protein

Single crystals of bovine P2 myelin protein have been grown in polyethylene glycol 4000 by the hanging-drop vapor diffusion method. Crystals belonging to space group P2(1)2(1)2(1) with cell dimensions a = 91.8 A, b = 99.5 A, c = 56.5 A (1 A = 0.1 nm). The diffraction pattern extends to better than 2.3 A resolution.     

Crystallization and preliminary X-ray diffraction studies of bovine recombinant immune interferon

Reproducible conditions have been established for the crystallization of recombinant bovine immune interferon. Two cystalline forms of this protein were obtained. A tetragonal form, space group P422, with unit cell dimensions a = b = 59.0 A and c = 125.7 A and an orthorhombic form, space group P2(1)2(1)2(1), with unit cell dimensions a = 42.80 A, b = 79.90 A and c = 85.64 A were obtained under similar crystallization conditions. The orthorhombic form diffracts to 2.6 A resolution, contains a single interferon dimer in the asymmetric unit of structure and is suitable for X-ray diffraction analysis.     

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 A resolution.     

Crystallization and crystal data on tyrosine phenol-lyase

Crystals of the apoenzyme of tyrosine phenol-lyase (EC 4.1.99.2), a pyridoxal 5'-phosphate-dependent enzyme from Citrobacter intermedius, have been grown by vapor diffusion of an ammonium sulfate solution to a protein solution. The crystals belong to space group P2(1)2(1)2, with dimensions of a = 75.5 A, b = 138.4 A and c = 94.1 A and diffract up to 2.7 A resolution. The asymmetric unit contains one half of the enzyme tetrameric molecule. Two heavy-atom derivatives of the crystals have been obtained.     

Preliminary crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.     

New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver

20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution. The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316. 0 A, and diffracts to 4.0 A resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.     

Crystallization and preliminary X-ray diffraction data of two crystal forms of bovine heart creatine kinase

Two crystal forms of bovine heart creatine kinase, which are suitable for X-ray diffraction studies, have been grown at room temperature using 2-methyl-2,4-pentanediol as the precipitant at pH 7.2. The space group of the orthorhombic form is P2(1)2(1)2, with unit cell dimensions a = 133 A, b = 128 A and c = 65 A, and there is one dimeric molecule in the asymmetric unit. The space group of the tetragonal form is P4(2)2(1)2, with unit cell dimensions a = b = 132 A and c = 75 A, with one subunit in the asymmetric unit. The tetragonal crystals diffract to at least 2.0 A resolution.     

Crystallization of and preliminary X-ray data for the plasma retinol-binding protein

Crystals of the human and rabbit plasma retinol-binding proteins have been grown from solutions of polyethylene glycol 6000 and CdCl2. Two crystal forms have been observed for the human protein, while the rabbit protein has only crystallized in one form which is isomorphous with one of the human serum retinol-binding protein crystals. The crystals differ in their morphologies, but are both in space group P212121 and have similar unit cell sizes (a = 45.9, b = 53.3, c = 72.0 A and a = 45.7, b = 48.7, and c = 76.5 A). The crystals diffract to approximately 2.0 A resolution. In both cases there is 1 molecule/asymmetric unit.     

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.     

Preliminary X-ray diffraction analysis of crystals of Bacillus thuringiensis toxin, a cell membrane disrupting protein

Crystals suitable for high resolution X-ray diffraction analysis have been reproducibly grown of the 24,000 Mr protein insect toxin from Bacillus thuringiensis. This protein, which demonstrates substantial insecticidal activity by inserting into phospholipid membranes, crystallizes as long square needles from polyethylene glycol 4000 at neutral pH. The crystals are of space group P4(1) and have cell dimensions of a = b = 33 A and c = 235 A, which suggests to us a predominantly helical motif for the protein's structure.     

Preliminary x-ray data on crystals of mandelate racemase

The recent development of a high-yield expression system and purification scheme for mandelate racemase has enabled us to produce sufficiently large quantities of pure enzyme to pursue x-ray crystallographic study. Large, single crystals of mandelate racemase have been grown from buffered polyethylene glycol (pH 8.0) in the presence of 10 mM magnesium chloride. The crystals grow in several habits, and we have identified two distinct tetragonal space groups in preliminary x-ray diffraction analysis. Crystals shaped as rectangular plates demonstrate 4/mmm Laue symmetry and systematic absences consistent with the space group I422. They have cell dimensions of a = b = 153 A and c = 181 A. Octahedrally shaped crystals of mandelate racemase display 4/m Laue symmetry and systematic absences consistent with the space group 14. Cell dimensions for these crystals are a = b = 113 A and c = 124 A. Based on estimates of Vm and on the measured density of the 1422 form, we suggest that two subunits of mandelate racemase (38,570 daltons/subunit) occupy the asymmetric unit in both crystal forms. Crystals of both forms diffract to beyond 3.0-A resolution. We are currently screening for isomorphous heavy-atom derivatives.     

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.     

Crystals and a low resolution structure of interleukin-2

Recombinant derived human interleukin-2 and an analog in which cysteine 125 has been replaced with alanine have been crystallized in a form suitable for x-ray diffraction. The crystals are triclinic, space group P1, with two protein molecules in the unit cell; unit cell parameters are a = 55.8 A, b = 40.1 A, c = 33.7 A, alpha = 90.0 degrees, beta = 109.3 degrees, gamma = 93.2 degrees. The interleukin-2 structure has been solved to 5.5 A resolution using heavy atom isomorphous replacement methods. The resultant low resolution model reveals a significant fraction of alpha helical secondary structure and outlines the overall tertiary structure of the molecule.     

Crystallization of isoelectrically homogeneous cholera toxin

Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. We have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. We have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by our procedure readily forms large single crystals. The crystal form (space group P2(1), a = 73.0 A, b = 92.2 A, c = 60.6 A, beta = 106.4 degrees, one molecule in the asymmetric unit) has been described previously [Sigler et al. (1977) Science (Washington, D.C.) 197, 1277-1278]. We have recorded data from native crystals of cholera toxin to 3.0-A resolution with our electronic area detectors. With these data, we have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. We are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.