Crystallization Of Proteinase Research Articles

High-resolution X-ray study of the effects of deuteration on crystal growth and the crystal structure of proteinase K.

Deuteration of macromolecules is an important technique in neutron protein crystallography. Solvent deuteration of protein crystals is carried out by replacing water (H(2)O) with heavy water (D(2)O) prior to neutron diffraction experiments in order to diminish background noise. The effects of solvent deuteration on the crystallization of proteinase K (PK) with polyethylene glycol as a precipitant were investigated using high-resolution X-ray crystallography. In previous studies, eight NO(3)(-) anions were included in the PK crystal unit cell grown in NaNO(3) solution. In this study, however, the PK crystal structure did not contain NO(3)(-) anions; consequently, distortions of amino acids arising from the presence of NO(3)(-) anions were avoided in the present crystal structures. High-resolution (1.1 Å) X-ray diffraction studies showed that the degradation of PK crystals induced by solvent deuteration was so small that this degradation would be negligible for the purpose of neutron protein crystallography experiments at medium resolution. Comparison of the nonhydrogen structures of nondeuterated and deuterated crystal structures demonstrated very small structural differences. Moreover, a positive correlation between the root-mean-squared differences and B factors indicated that no systematic difference existed.

Crystallization of proteinase K complexed with substrate analogue peptides on US space missions STS-91 and STS-95

Crystals of proteinase K in complex with synthetic substrate analogues have been grown under microgravity on the US space shuttle missions STS-91 and STS-95 using the vapor diffusion apparatus (c-VDA) supplied by the Center for Macromolecular Crystallography at the University of Alabama at Birmingham. The crystals obtained under microgravity are compared with those grown simultanously on ground in identical c-VDA reactors and in conventional hanging-drop set-ups. The diffraction quality of space- and ground-grown crystals has been assessed by collecting complete data sets with a conventional X-ray source and with synchrotron radiation. Crystals grown in microgravity are clearly superior to those grown in the identical hardware on earth in terms of crystal habit and diffraction power. In comparison to best terrestrial crystals obtained in conventional hanging-drop set-ups the differences in crystal size and diffraction quality are less, but still confirm the benefit of microgravity for the crystallization of proteinase K–substrate analogue complexes.

Phase diagram of crystallization of Aspergillus niger acid proteinase A, a non-pepsin-type acid proteinase

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase with an extremely low isoelectric point (pI 3.3). The protein is crystallized from ammonium sulfate solutions of pH lower than 4. The crystallization is affected by the presence of dimethylsulfoxide (DMSO). We have studied the phase diagram of the crystallization of proteinase A in the absence and presence of DMSO, to clarify crystallization at such an extremely low pH and to study the effects of DMSO. The results indicate that the logarithm of protein solubility is a rectilinear function of ammonium sulfate concentration in both the absence and presence of DMSO. DMSO definitely lowers the solubility at relatively low concentrations of ammonium sulfate, but had little effect on protein solubility at higher concentrations of ammonium sulfate.

Isolation, biochemical characterization and crystallization of the p15 gag proteinase of myeloblastosis associated virus expressed in E. coli

1. 1. The p15 gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E. coli expression system. The massive expression of the intentionally truncated precursor (pr25 lac-Δgag ) was accompanied by its structurally correct processing. 2. 2. Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed. 3. 3. The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N-terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates. The identity of both enzymes was shown. 4. 3. Contrary to reported data, the amino acid sequence of the p15 gag proteinase diners from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing. 5. 4. Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well at 2.3 Å without deterioration.

Crystallization and preliminary X-ray investigation of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase distinctly different in various properties from the family of pepsin-type aspartic proteinases, and so far it remains unknown which residues participate in the catalysis of the enzyme and how the mechanism operates. The acid proteinase A was crystallized from an ammonium sulfate solution by the hanging-drop vapor diffusion method. The space group of the crystals was P2 12 12 1 with unit cell dimensions of a = 54·7 A ̊ , b = 70·4 A ̊ and c = 38·0 A ̊ . On the assumption that there is one enzyme molecule in the asymmetric unit, the calculated ratio of volume to unit protein mass ( V m) was 1·64 Å 3 per dalton. Diffraction data were collected up to a resolution higher than 1·5 Å, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. The crystal of proteinase A is, therefore, suitable for the structural analysis with a high resolution.

幽門垂蛋白分解酵素に関する研究-I

The purification of the proteinase of tunny pyloric caeca was carried out as follows; the distilled water extraction of ground material, salting-out with ammonium sulphate, dialysis, rivanol treatment for removal of amylase, fuller's earth treatment, and precipitation and cry-stallization with acetone (Fig. 3). The crystals of proteinase were obtained in the form of needles (Fig. 4). The crystalline proteinase was obtained with an yield of 1.5% in activity and a specific activity increased 20.6 times as compared with the original solution (Table 3).