Crystallization of a chymotrypsin inhibitor from Erythrina caffra seeds

Crystals of a chymotrypsin inhibitor from Erythrina caffra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 67.2 A and alpha = 99.4 degrees, and diffract to 3 A resolution.     

In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein

Winged bean chymotrypsin inhibitor (WCI) has an intruding residue Asn14 that plays a crucial role in stabilizing the reactive site loop conformation. This residue is found to be conserved in the Kunitz (STI) family of serine protease inhibitors. To understand the contribution of this scaffolding residue on the stability of the reactive site loop, it was mutated in silico to Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD) simulations were carried out on the mutants. The results of MD simulations reveal the conformational variability and range of motions possible for the reactive site loop of different mutants. The N-terminus side of the scissile bond, which is close to a beta-barrel, is conformationally less variable, while the C-terminus side, which is relatively far from any such secondary structural element, is more variable and needs stability through hydrogen-bonding interactions. The simulated structures of WCI and the mutants were docked in the peptide-binding groove of the cognate enzyme chymotrypsin and the ability to form standard hydrogen-bonding interactions at P3, P1 and P2' residues were compared. The results of the MD simulations coupled with docking studies indicate that hydrophobic residues like Leu and Val at the 14th position are disruptive for the integrity of the reactive site loop, whereas a residue like Thr, which can stabilize the C-terminus side of the scissile bond, can be predicted at this position. However, the size and charge of the Asn residue made it most suitable for the best maintenance of the integrity of the reactive site loop, explaining its conserved nature in the family.     

Crystallization and preliminary studies of lima bean trypsin inhibitor

Crystals of lima bean trypsin inhibitor (LBTI) were obtained by using the vapor phase equilibration technique with sodium/potassium tartrate as the precipitating agent. The space group was determined to be cubic, I2₁3 with a= 110.2 Å. These crystals diffract to about 1.9 Å resolution. Preliminary analysis of self-rotation maps (calculated from native x-ray intensity data) suggests the presence of two monomers in the asymmetric unit. LBTI is very thermostable and retains activity even after boiling for 10 minutes. This property is exploited as part of its purification procedure. © 1997 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of coxsackievirus B1.

Preparations of coxsackievirus B1 (CVB1) derived from an infectious cDNA clone have been crystallized in multiple crystal forms. Using high intensity synchrotron radiation, an orthorhombic form of the crystals was shown to diffract X-rays to at least 2.9 A resolution. The unit cell has a primitive lattice with dimensions a = 323 A, b = 450 A, and c = 522 A. A crystallographic asymmetric unit of these CVB1 crystals probably contains an entire virus particle, implying the presence of 60-fold non-crystallographic redundancy. This CVB1 crystal form appears to be suitable for high-resolution structure determination by X-ray crystallography.     

Crystallization and preliminary X-ray studies of two isolectins from the seeds of Lathyrus ochrus

Two isolectins from the seeds of Lathyrus ochrus, LOL I and LOL II, which specifically bind N-acetyllactosamine, have been crystallized using the hanging-drop method and the interface diffusion method, respectively. In the case of LOL I, 2-methylpentane-2,4-diol, polyethylene-glycol 400 or ammonium sulphate have been used as precipitating agents. The best crystals of LOL I were grown at room temperature from a solution of 40% (v/v) methylpentane diol, 50 mM-Hepes at pH 7.5. LOL II crystals have been grown at room temperature from a solution of 32% (v/v) methylpentane diol, 50 mM-2-(N-morpholino)-ethanesulphonic acid at pH 5.5. X-ray examination of the LOL I and LOL II crystals shows that both are monoclinic, space group P2(1). Their cell dimensions are: LOL I, a = 56.4 A, b = 138.8 A, c = 62.9 A, beta = 91 degrees; and LOL II, a = 54.8 A, b = 71.4 A, c = 105.5 A, beta = 105 degrees. Density measurements of the crystals of LOL I indicate that there are two molecules per asymetric unit (Vm = 2.07 A3/dalton). LOL I crystals diffract strongly up to at least 1.8 resolution. Putative crystals of complexes of LOL I with various glycosides were obtained through co-crystallization under the conditions used for the native protein.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B

The Δ⁵-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2₁2₁2₁ with unit cell dimensions of a = 36.48 Å, b = 74.30 Å, c = 96.02 Å, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Å resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.     

Analysis of differential accumulation of winged bean Kunitz chymotrypsin inhibitor mRNA species by a sequence-specific termination method

Winged bean Kunitz chymotrypsin inhibitor (WCI) is encoded by a multigene family and accumulation of its mRNA is restricted in mid-maturation stage seeds and tuberous roots. In this paper, we analyzed the accumulation of mRNA derived from each WCI gene using a novel method: sequence-specific termination analysis. The results demonstrated that the accumulation of each WCI mRNA was differentially regulated in winged bean plants.     

Conformation and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC)

Spectrophotometric measurement was found to be a sensitive method for evaluating the stability of the chymotrypsin inhibitor from the winged bean. The thermal stability of this protein in aqueous solution was much greater at pH 3 than at pH 8 or pH 11. Evidence from u.v. absorption and from circular dichroism indicated that irreversible conformation changes occurred at higher temperature (greater than 70 degrees). Circular dichroism and optical rotatory dispersion studies at pH 8 show that the inhibitor is rich in beta-structure and virtually devoid of alpha-helix in aqueous solution. We conclude from experiments with denaturing solvents that the inhibitor is very stable and that high concentrations of denaturant are required before unfolding occurs. Chemical modification experiments with tetranitromethane were consistent with a tight stable structure; even in 6M guanidine hydrochloride only three of the five tyrosine residues in the inhibitor molecule were nitrated. However, tyrosine does not seem to be implicated at the reactive site of the inhibitor. Interaction of the inhibitor with alpha-chymotrypsin and chymotrypsin B was also followed by difference spectroscopy in the ultraviolet region. Difference spectra were detected that were characteristic of changes in the environment of both tyrosine and tryptophan chromophores. Comparison of the spectral data obtained for the interaction of the inhibitor with bovine alpha-chymotrypsin and with chymotrypsin B indicated that a tryptophan residue may be involved at the reactive site of the inhibitor. Spectral changes were also detected for the interaction between the chymotrypsin inhibitor and trypsin, although it is well established that the specificity of this inhibitor is restricted to the chymotrypsins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L) Dc)

The specificity of the winged bean chymotrypsin inhibitor is restricted to the chymotrypsins (EC 3.4.21.1 and EC 3.4.21.2). Trypsins (EC 3.4.21.4), elastase (EC 3.4.21.11), subtilisins (EC 3.4.21.14), proteinase K (EC 3.4.21.14) and Pronase (EC 3.4.24.4) are not inhibited. The inhibitor reacts with two molecules of chymotrypsin to form a stable complex (Mr approx. 70 0000) which was isolated by gel filtration on Sephadex G-100. When mixed with substrate, the interaction of the inhibitor with alpha-chymotrypsin is characterized by substrate-induced dissociation of the complex. In contrast, the interaction with chymotrypsin B is quantitative with no substrate-induced dissociation. The inhibitor reacts with alpha-chymotrypsin to form a 1 : 2 molar complex at all ratios of [I]/[E]; however, the interaction with chymotrypsin B is characterized by the formation of initially of a 1 : 1 molar complex at [I] greater than [E] followed by the formation of the 1 : 2 molar complex at [I] less than 2[E]; an intermediate species of Mr approx. 48 000 was demonstrated by gel filtration on Sephadex G-100. The inhibitor is stable over the pH range 2.0-11.5 and to heating up to 70 degrees C at pH 4.1 and 8.0, and up to 90 degrees C at pH 3.0. The inhibitor resists denaturation in 8.0 M urea at pH 8.0 and 4.0, is stable in 0.12 M beta-mercaptoethanol at pH 8.0; however, reduction in 8.0 M urea results in a loss of inhibitory activity. The inhibitor resists digestion with pepsin at pH 2.0, being only slowly degraded over a period of 7 days with an equimolar amount of pepsin.     

Purification,crystallization and preliminary crystallographic studies of a class II chitinase from kidney bean seeds

A new member of class II chitinase from Phaseolus vulgaris was purified and crystallized. Diffraction data to 2.7A resolution have been collected and the preliminary crystallographic studies have been completed. The space group is P1 with unit cell parameters of a=36.32A, b=46.24A, c=70.36A, a =97.9 degrees, b=103.8 degrees and g =110.5 degrees. Molecular replacement and initial refinement statistics indicate there are two chitinase molecules in the crystallographic asymmetric unit.     

Crystallization and preliminary X-ray diffraction studies of C-phycocyanin from a red alga, Porphyra tenera

C-Phycocyanin from a red alga, Porphyra tenera, has been crystallized by the vapor-diffusion procedure. Both orthorhombic and hexagonal forms were obtained from ammonium sulfate solutions, whereas only the orthohombic form was selectively grown from sodium citrate solutions. The orthorhombic crystals are more suitable for further crystallographic work; their space group is P2(1)2(1)2(1), with unit-cell dimensions of a = 105, b = 121, and c = 184 A. The asymmetric unit comprises two (alpha beta)3 trimer molecules of C-phycocyanin. These crystals diffract X-rays up to about 3 A resolution.     

Crystallization and preliminary X-ray diffraction studies of intact EF-Tu from Thermus aquaticus YT-1

Many attempts have been made to elucidate the three-dimensional structure from elongation factor Tu, but so far the only crystals suitable for X-ray crystallography contained a partially degraded protein. Here, we report the crystallization of a fully active, intact EF-Tu from thermus aquaticus. The crystals belong to hexagonal space group P6(3)(22) and diffract up to 2.6 A. The cell dimensions are a = b = 178 A, c = 238 A and 6 molecules are contained per asymmetric unit.     

Crystallization and preliminary X-ray study of the cathepsin B complexed with CA074, a selective inhibitor.

Cathepsin B from bovine spleen has been purified and crystallized as a complex with a specific inhibitor CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L- isoleucyl-L-proline], using the hanging-drop method. The complex crystals obtained from 50 mM-citrate buffer (pH 3.5) belong to the tetragonal space group P4(1) (or P4(3)) with a = 73.06 A and c = 141.59 A, and diffract beyond 2.2 A resolution. There are two complex molecules per asymmetric unit giving a packing density of 3.37 A3/Da and indicating a high solvent content of 63.5%.     

Crystallization and preliminary X-ray crystallographic study of the leech protease inhibitor guamerin and its complex with bovine pancreatic chymotrypsin

Guamerin, a small peptide inhibitor of the serine protease from Hirudo nipponia, was expressed in yeast and crystallized using the vapor diffusion method, with MPD as precipitant. The crystal was found to belong to the monoclinic P2(1) space group with unit cell parameters a=136.06, b=206.59, c=227.39 A, beta=105.03 degrees. The guamerin/bovine pancreatic chymotrypsin complex was also crystallized using PEG 8K as precipitant. The space group was identified as P2(1)2(1)2(1) with unit cell parameters of a=44.01, b=44.30, c=122.47 A. The diffraction data of the complex were collected up to a resolution of 2.4 A using a synchrotron-radiation source under cryogenic condition.     

Crystallization and preliminary X-ray diffraction studies of a peroxidase from barley grain.

Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P2(1)2(1)2(1) with unit cell dimensions a = 62.95 A, b = 66.24 A and c = 70.78 A. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 A.     

Crystallization and preliminary X-ray diffraction studies of peroxidase from a fungus Arthromyces ramosus

Peroxidase (donor: H₂O₂ oxi-doreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P4₂2₁2, with unit cell dimensions of a = b = 74.5 Å, c = 117.6 Å. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 Å resolution and are stable to X-rays. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of mandelonitrile lyase from almonds

Single crystals of three different isoenzymes of (R)?(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2_l with unit cell parameters a = 69.9, b = 95.1, c = 95.6 Å, and β = 118.5°. A complete set of diffraction data has been collected to 2.6 Å resolution on native crystals of isoenzyme III. © 1994 Wiley-Liss, Inc.