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.

Immune responses against recombinant tick antigen, Bm95, for the control of Rhipicephalus (Boophilus) microplus ticks in cattle.

Immune responses against Bm95 recombinant cattle tick antigen and its protective efficacy for control of Rhipicephalus (Boophilus) microplus ticks were determined in experimental crossbred cow calves. Anti-Bm95 antibody titers, as assessed by indirect ELISA, in immunized calves ranged from 196.1+/-13.7 on day 0 to 7979.9+/-312.5 on day 110 post-primary immunization. The rise in antibody titer was statistically significant (p<0.01) throughout the study period. Besides this, constantly higher lymphoproliferative response (LPR), as assessed by lymphocyte stimulation test, was observed from 10 days post-immunization, but a positive LPR of antigen stimulated cells in immunized animals was recorded only on day 50 and day 70 post-immunization. Following challenge of immunized calves with larvae of R. microplus, significant increase (p<0.01) in rejection percentage, mean number of damaged ticks, mean percentage of dead ticks, and decrease in engorgement weight were recorded in immunized animals. Also, there were significant differences (p<0.01) in preoviposition period, oviposition period, egg mass weight and percent hatchability between the immunized and control calves. The percent reduction in number of adult females in vaccinated calves, reduction in mean weight of egg masses, percent reduction in mean weight and reduction in fertility of engorged females collected from vaccinated calves were determined and the efficacy of Bm95 recombinant cattle tick antigen was 81.27%.

Tertiary structures, receptor binding, and antigenicity of insulinlike growth factors.

Three-dimensional models for human insulinlike growth factors (IGF-I and IGF-II) have been constructed by using interactive molecular graphics. It is suggested that the two growth factors have structures in which the A and B chains and the hydrophobic cores are identical to those of insulin. The conformations of the connecting peptides and COOH-terminal extensions are predicted by statistical methods but the structures are limited by the constraints implied by the insulinlike part. The models explain the nonsuppressibility by anti-insulin antibodies of the IGFs and show that part of the insulin receptor-binding region is conserved, which explains the growth factors' ability to bind insulin receptors.

Polypeptide hormone-receptor interactions: the structure and receptor binding of insulin and glucagon.

Insulin is a small globular protein with a well defined tertiary structure which is closely similar in all species with the exception of certain hystricomorphs such as the guinea pig. Insulin-like growth factor is homologous with insulin and probably has an insulin-like tertiary structure. In contrast glucagon is not a globular protein. It exists as an equilibrium population of conformers with low helix content at physiological concentrations but attains a largely helical conformation on association to trimers. The receptor binding of insulin depends critically on the correct three-dimensional juxtaposition of groups (A1, A21, B25, etc) and involves both hydrophobic and polar interactions. In insulin-like growth factor part of the insulin receptor region is thought to be buried in extra peptide, so explaining its weak binding to insulin receptors. In contrast the glucagon receptor complex probably involves largely hydrophobic contacts which are possible when a helical conformer is formed.

Insulin-like growth factor: a model for tertiary structure accounting for immunoreactivity and receptor binding.

A model for the three-dimensional structure of insulin-like growth factor (IGF) is proposed based on the close sequence homology of IGF with insulin, the tertiary structure of which is known. The IGF molecule is postulated to have an insulin-like main chain conformation for residues equivalent to B6--B27 and A1--A21 and a hydrophobic core nearly identical to that of insulin. A short connecting peptide of twelve residues and an extension at the COOH-terminus are easily accommodated on the molecular surface. The surface involved in dimer formation in insulin is largely conserved, but the zinc-binding histidine and many residues involving hexamerization are very different from those of insulin and it is unlikely that IGF forms zinc hexamers. The model provides a ready explanation for the inability of IGF to bind antibodies to insulin and for its ability to bind insulin receptors with low affinity.

The subunit structure of human macrophage migration inhibitory factor: evidence for a trimer.

The subunit structure of human macrophage migration inhibitory factor (MIF) has been studied by preliminary X-ray analysis of wild-type and selenomethionine-MIF and dynamic light scattering. Crystal form I of MIF belongs to space group P2(1)2(1)2(1) and is grown from 2 M ammonium sulfate at pH 8.5. A native data set has been collected to 2.4 A resolution. Self-rotation studies and Van values indicate that three molecules per asymmetric unit are present. A data set to 2.8 A resolution has been collected for crystal form II, which belongs to space group P3(1)21 or P3(2)21 and grows from 2 M ammonium sulfate, 2% polyethylene glycol (average molecular mass 400) 0.1 M HEPES, pH 7.5. Three, four, five or six monomers in the asymmetric unit are consistent with Van values for this crystal form. Analysis of crystal form II containing selenomethionine-MIF indicates nine selenium sites are present per asymmetric unit. Dynamic light scattering of MIF suggests that the major form of the protein in solution is a trimer. The results of these studies are in contrast to previous reports indicating that MIF is a monomer or dimer. The subunit arrangement of MIF is similar to that of tumor necrosis factor and suggests that signal transduction might require trimerization of receptor subunits.

The crystal structures of [Met5]enkephalin and a third form of [Leu5]enkephalin: observations of a novel pleated beta-sheet.

The structures of [Met5]enkephalin (Tyr-Gly-Gly-Phe-Met) and [Leu5]enkephalin (Tyr-Gly-Gly-Phe-Leu) have been determined from single crystal x-ray diffraction data and refined to residuals of 0.100 and 0.092, respectively. The [Met5]enkephalin structure consists of dimers forming antiparallel beta-sheets extending in the monoclinic ac plane with 10.6 water molecules per dimer. The two molecules, related by pseudo two-fold axes, have similar backbone conformations and similar tyrosine and phenylalanine side-chain conformations. Both methionine residues are disordered and the disorder is different in the two independent molecules. Additional hydrogen bonds connect adjacent dimers to form infinite sheets normal to the b axis. The water molecules are found mainly in the interstices between the sheets. [Leu5]Enkephalin crystallizes as a monohydrate that is isomorphous with the [Met5]enkephalin structure with respect to the beta-sheet but different with respect to the tyrosine and phenylalanine side-chain conformations and water content. The peptide chains in both structures are fully extended and more nearly planar than pleated. The planes of the peptide chains in the dimers form an angle of 143.3 degrees with one another in [Met5]enkephalin and 156.0 degrees in [Leu5]enkephalin. This produces a zigzag pattern or pleat in the beta-sheets perpendicular to the direction of the peptide chains and, therefore, perpendicular to the normal beta-sheet pleat. The average repeat distance between Ni and Ni+2 in the peptide chains of both structures is 7.10 A, versus an ideal value of 6.68 A.