Form Of Bovine Pancreatic Ribonuclease Research Articles

Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering.

The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.

New monoclinic form of bovine pancreatic ribonuclease A from a salt solution and comparison of intermolecular interactions in ribonucleases A

A new monoclinic form of bovine pancreatic ribonuclease A was obtained together with the known trigonal form using sodium chloride and ammonium sulfate as precipitants. The new monoclinic form was stable at high salt concentrations. Crystal structure analysis of the new monoclinic form was carried out, and the intermolecular interactions were compared with those of the trigonal form. In the new monoclinic form, there were two pairs of non-crystallographic dimers, both of which were almost equivalent to the dimer in the trigonal form. Molecular layers were formed by the dimers, and a water region was spread between the molecular layers. Although the overall crystal structures of the two crystal forms are quite different, common successive interaction between the dimers aligned along the two-fold screw axis. Analyses of the contact surfaces were carried out, together with the other crystal forms of native ribonuclease A, resulting in a qualitative understanding of the relationship between intermolecular interactions and the crystallization conditions.

Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped dimeric form (NCD-BS), which presents a compact structure that allows the molecule to escape ribonuclease inhibitor (RI). A key role in the acquisition of this structure has been attributed to the concomitant presence of a proline in position 19 and a leucine in position 28. The introduction of Leu28, Cys31, and Cys32 and, in addition, of Pro19 in the sequence of bovine pancreatic ribonuclease (RNase A) has produced two dimeric variants LCC and PLCC, which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase. The crystal structure analysis of the noncovalent swapped form (NCD) of LCC and PLCC, complexed with the substrate analogue 2 '-deoxycytidylyl(3 ',5 ')-2 '-deoxyguanosine, has revealed that, differently from NCD-BS, the dimers adopt an opened quaternary structure, with the two Leu residues fully exposed to the solvent, that does not hinder the binding of RI. Similar results have been obtained for a third mutant of the pancreatic enzyme, engineered with the hinge peptide sequence of the seminal enzyme (residues 16-22) and the two cysteines in position 31 and 32, but lacking the hydrophobic Leu residue in position 28. The comparison of these three structures with those previously reported for other ribonuclease swapped dimers strongly suggests that, in addition to Pro19 and Leu28, the presence of a glycine at the N-terminal end of the hinge peptide is also important to push the swapped form of RNase A dimer into the compact quaternary organization observed for NCD-BS.

Selective stabilization of a labile mutant form of bovine pancreatic ribonuclease A by antibodies

The region between the amino acids 31-46 was previously identified as being first exposed during thermal unfolding of bovine pancreatic ribonuclease A (RNase). The exchange of one amino acid (Leu35toSer) in this unfolded region of RNase is shown to have a dramatic destabilizing effect (ΔTm=9 °C). Antibodies raised against a peptide corresponding to the sequence of the labile region, S32-V43, of RNase were effective in stabilizing L35S-RNase against thermal inactivation (65 °C for 2 h) and surpassed the stabilization effect of antiRNase antibodies. An 11% contribution to the stabilizing effect of antiRNase antibodies resulted from antibodies recognizing the unfolding region of the enzyme.

The ultrahigh resolution crystal structure of ribonuclease A containing an isoaspartyl residue: hydration and sterochemical analysis

Crystals of the deamidated form of bovine pancreatic ribonuclease which contains an isoaspartyl residue in position 67 diffract to 0.87 Å at 100 K. We have refined the crystallographic model using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.101 for all observed reflections in the resolution range 61.0–0.87 Å. The ratio observations/parameters is 7.2 for the final model. This structure represents one of the highest resolution protein structures to date and interestingly, it is the only example containing more than one molecule in the asymmetric unit with a resolution better than 1.0 Å. The non-crystallographic symmetry has been used as a validation check of the geometrical parameters and it has allowed an estimate for an upper limit of errors associated with this high resolution model. In the present structure it was possible to obtain a more accurate picture of the active site whose electron density was not clearly interpretable in the previous 1.9 Å resolution structure. In particular, the P1 site is alternatively occupied either by a sulphate anion or by a water molecule network. Most of hydrogen atoms were visible in the electron density maps, including those involved in C α—H α···O interactions. Analysis of protein-solvent interactions has revealed the occurrence of an extensive cluster of water molecules, predominantly arranged in pentagonal fused rings and surrounding hydrophobic moiety of side-chains. Finally, in spite of the limited sample of residues, we have detected a clear dependence of backbone N—C α—C angle on residue conformation. This correlation can be fruitfully used as a valuable tool in protein structure validation.

N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin.

Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes.

The crystal structure of ribonuclease B at 2.5-A resolution.

The glycosylated form of bovine pancreatic ribonuclease, RNase B, was crystallized from polyethylene glycol 4000 at low ionic strength in space group C2 with unit cell dimensions of a = 101.81 A, b = 33.36 A, c = 73.60 A, and beta = 90.4 degrees. The crystals, which contained two independent molecules of RNase B as the asymmetric unit, were solved by a combination of multiple isomorphous replacement and molecular replacement approaches. The structures of the two molecules were refined to 2.5-A resolution and a conventional R factor of 0.22 using a constrained-restrained least squares procedure (CORELS). Complexes were also investigated of RNase B plus ruthenium pentaamine and between RNase B and a substrate analogue iodouridine. The polypeptide backbones of the two molecules of RNase B in the asymmetric unit were found to be statistically identical and their differences from RNase A to be statistically insignificant. The carbohydrate chains of both molecules extended into solvent cavities in the crystal lattice and appear to be disordered for the most part. The oligosaccharides appear to exert no influence on the structure of the protein. Iodouridine was observed to bind identically in the pyrimidine site of both RNase B molecules and in a way apparently the same as that previously observed for RNase A. Ruthenium pentaamine bound at histidine 105 of both RNase B molecules in the asymmetric unit, but at a number of secondary sites as well. An array of bound ions was observed by Fo-Fc difference Fourier syntheses. These ions were proximal to lysine and arginine residues at the surface of the proteins while a pair of strong ion binding sites were seen to fall exactly in the active site clefts of both RNase B molecules in the asymmetric unit.

Water-mediated transformations in protein crystals

Different crystal forms of bovine pancreatic ribonuclease A and hen egg white lysozyme, 2Zn insulin, 4Zn insulin and crystals of concanavalin A were examined under controlled environmental humidity in the relative humidity (r.h.) range of 100 to 75%. Many of them, but not all, undergo reversible structural transformations as evidenced by discontinuous changes in the diffraction pattern, the unit-cell dimensions and the solvent content. Tetragonal, orthorhombic and monoclinic lysozyme and a new crystal form of ribonuclease A show transformations at r.h.'s above 90%. Monoclinic lysozyme transforms at low r.h. to another monoclinic form with nearly half the original cell volume. The well known monoclinic form of ribonuclease A grown from aqueous ethanol solution undergoes two transformations while the same form grown from 2-methyl-2,4pentanediol (MPD) solution in phosphate buffer does not transform at all. Soaking experiments involving alcohol solutions demonstrate that MPD has the effect of decreasing the r.h. at which the transformation occurs. Triclinic lysozyme, 2Zn insulin, 4Zn insulin and the crystals of cancanavalin A do not transform in the 100 to 75% r.h. range before losing crystallinity. The results obtained so far indicate that the crystal structure has a definite influence on water-mediated transformations. The transformations do not appear to depend critically on the amount of solvent in the crystals but the r.h. at which they occur is influenced by the composition of the solvent. The transformations appear to involve changes in crystal packing as well as conformational transitions in protein molecules. The present investigations and other related studies suggest that water-mediated transformations in protein crystals could be very useful in

Structural transformations in protein crystals caused by controlled dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them.

Crystallization and preliminary x-ray data for a glycosylated form of bovine pancreatic ribonuclease.

A glycosylated form of bovine pancreatic ribonuclease, ribonuclease B, has been crystallized in a form suitable for three-dimensional x-ray structure determination. Crystals grown from polyethylene glycol solutions display high resolution diffraction patterns indicating the orthorhombic space group P2(1)2(1)2(1) with a = 59.2 (+/- 0.1), b = 56.0 (+/- 0.1), and c = 81.0 (+/- 0.2). A, V = 2.7 x 10(5) A3. With 8 molecules/unit cell Vm = 2.28 A3/dalton. The systemically weak intensity of all reflections with their l indices odd suggests the likely alignment of a noncrystallographic 2-fold axis near the crystal c axis.

Crystalline forms of bovine pancreatic ribonuclease. Some new modifications

Crystalline forms of bovine pancreatic ribonuclease. Some new modifications

On the thermal melting and recrystallization of ribonuclease crystals and the heterogeneity of ribonuclease

Various crystalline forms of bovine pancreatic ribonuclease, heated in their mother liquors, melt to form gels at temperatures characteristic of the crystal forms used. On incubation at 25° C, crystals grow again in the samples, usually of the same form as had originally been melted. Indications were obtained of a type of heterogeneity other than that previously reported in the literature for this protein.

Crystalline forms of bovine pancreatic ribonuclease: techniques of preparation, unit cells, and space groups

Crystalline forms of bovine pancreatic ribonuclease: techniques of preparation, unit cells, and space groups