1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.     

Effects of neutral salts on the circular dichroism spectra of ribonuclease a and ribonuclease s

The circular dichroism (CD) spectra of ribonuclease A, ribonuclease S, and N-acetyltyrosineamide were recorded as a function of pH in the presence of various concentrations of inorganic salts. Above pH 9.0 salting-in of tyrosine residues increases their intramolecular associations. This association enhances the contribution from these residues to the CD spectrum leading to an apparent titration curve that is shifted toward lower pH. The data indicate that unfolding of ribonuclease A and S by inorganic salts does not begin with disrupting existing electrostatic interactions. But, as the unfolding process progresses, disruption of electrostatic interactions may take place. This is consistent with our previous calorimetric studies which suggest that unfolding of ribonuclease A by salts proceeds initially by energetically favorable solvation of the folded protein. An increase in ellipiticity at 275 nm of partially unfolded protein in salt was observed as the pH was changed from 7.0 to 4.0. This observation may suggest that the isothermal unfolding of the protein by salts at low pH proceeds through an intermediate step which involves histidine residues and causes a conformational change in the tyrosine's asymmetric environment.     

A study on the positive ellipticity in the circular dichroism of ribonuclease A

The small positive elliplicity near 239 nm in the CD spectrum of RNase has been investigated as a function of pH. Theoretical calculations using CD parameters representing buried or exposed tyrosine residues have been carried out. A comparison of the theoretical calculations with experimental data suggests that the changes in the band's intensity, as a function of pH, arise mainly from electronic transitions associated with the tyrosine residues. The buried tyrosine residues are the major contributors to the ellipticity in this region at neutral pH. At higher pH contributions from exposed residurs are also observed.     

The compactness of ribonuclease A and reduced ribonuclease A

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R_g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R_g value of about 17.3 Å in 8 M urea. The reduced ribonuclease A is more expanded, its R_g value is about 20 Å in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.     

Role of unique basic residues of human pancreatic ribonuclease in its catalysis and structural stability

Human pancreatic ribonuclease (HPR) and bovine RNase A belong to the RNase A superfamily and possess similar key structural and catalytic residues. Compared to RNase A, HPR has six extra non-catalytic basic residues and high double-stranded RNA (dsRNA) cleavage activity. We mutated four of these basic residues, K6, R32, K62, and K74 to alanine and characterized the variants for function and stability. Only the variant K74A had an altered secondary structure. Whereas R32A and K62A had full catalytic activity, the mutants K6A and K74A had reduced activity on both ssRNA and dsRNA. The mutations of K62 and K74 resulted in reduction in protein stability and DNA double helix unwinding activity of HPR; while substitutions of K6 and R32 did not affect either the stability or helix unwinding activity. The reduced catalytic and DNA melting activities of K74A mutant appear to be an outcome of its altered secondary structure. The basic residues studied here, appear to contribute to the overall stability, folding, and general catalytic activity of HPR.     

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.     

Compensating effects on the cytotoxicity of ribonuclease A variants

Ribonuclease (RNase) A can be endowed with cytotoxic activity by enabling it to evade the cytosolic ribonuclease inhibitor protein (RI). Enhancing its conformational stability can increase further its cytotoxicity. Herein, the A4C/K41R/G88R/V118C variant of RNase A was created to integrate four individual changes that greatly decrease RI affinity (K41R/G88R) and increase conformational stability (A4C/V118C). Yet, the variant suffers a decrease in ribonucleolytic activity and is only as potent a cytotoxin as its precursors. Thus, individual changes that increase cytotoxicity can have offsetting consequences. Overall, cytotoxicity correlates well with the maintenance of ribonucleolytic activity in the presence of RI. The parameter (k(cat)/K(m))(cyto), which reports on the ability of a ribonuclease to manifest its ribonucleolytic activity in the cytosol, is especially useful in predicting the cytotoxicity of an RNase A variant.     

A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.     

Primary structure of pronghorn pancreatic ribonuclease: Close relationship between giraffe and pronghorn

Summary Pancreatic ribonuclease from pronghorn (Antilocapra americana) was isolated and its amino acid sequence was determined from a tryptic digest of the performic acid-oxidized protein. Peptides were positioned by homology with other ribonucleases. Only peptides that differed in amino acid composition from the corresponding peptides of ox or goat ribonucleases were sequenced.In a most parsimonious tree of pancreatic ribonucleases, pronghorn and giraffe were placed together and these two were placed with the bovids, leaving the deer as a taxon separate from the other ruminants. The amino acid replacements that determine this tree topology are three rarely occurring replacements shared by pronghorn and giraffe. Notwithstanding their close phylogenetic relationship, both ribonucleases differ strongly in extent of glycosidation, net charge and antigenic properties.     

Sequential 1H-NMR assignment and solution structure of bovine pancreatic ribonuclease A

Assignments for 1H-NMR resonances of most of the residues of bovine pancreatic ribonuclease (RNase A) have been obtained by sequence-specific methods. Identification and classification of spin systems have been carried out by two-dimensional phase-sensitive correlated spectroscopy (360 MHz) and single relayed coherence transfer spectroscopy. Sequence-specific assignments have been achieved by phase-sensitive two-dimensional nuclear Overhauser effect spectroscopy. To overcome the problem of spectral overlap use has been made of (a) an exhaustive analysis of partly exchanged RNase A (spectra in D2O), (b) a comparison with the subtilisin-modified enzyme (RNase S) and (c) small spectral perturbations caused by changes in pH and temperature. The secondary structure elements have been identified from the observed sequential, medium and long-range nuclear Overhauser effects together with data from amide-exchange rates. All information collected leads to the conclusion that the crystal and the solution structures are closely similar.     

An evidence for the equilibrium unfolding intermediates of ribonuclease A by tritium labeling method

A formation of a molten globule in the unfolding of ribonuclease A could be considered as an evidence supporting a hypothesis on the existence of such intermediates on the pathway of a protein folding. Using a novel technique (tritium labeling method) we have showed that the ribonuclease A equilibrium unfolding in urea and guanidinium chloride (GuCl) solutions proceeds through a formation of intermediates whose properties (compactness, retention of the larger part hydrophobic core, secondary structure, and native-like folding pattern) correspond to the fundamental characteristics of the molten globule state. The both intermediates are the “wet” molten globules (the globule interior contains the water molecules). The results reveal the noticeable distinctions in intermediates structure, first of all, in the extent of their compactness. The urea intermediate is less compact than that in GuCl. It is shown that the refolding of the protein denatured by GuCl results in the formation of the intermediate which enzyme activity is virtually the same as the activity of the native protein.     

Differences in the denaturation behavior of ribonuclease A induced by temperature and guanidine hydrochloride

Moderate temperatures or low concentrations of denaturants diminish the catalytic activity of some enzymes before spectroscopic methods indicate protein unfolding. To discriminate between possible reasons for the inactivation of ribonuclease A, we investigated the influence of temperature and guanidine hydrochloride on its proteolytic susceptibility to proteinase K by determining the proteolytic rate constants and fragment patterns. The results were related to changes of activity and spectroscopic properties of ribonuclease A. With thermal denaturation, the changes in activity and in the rate constants of proteolytic degradation coincide and occur slightly before the spectroscopically observable transition. In the case of guanidine hydrochloride-induced denaturation, however, proteolytic resistance of ribonuclease A initially increases accompanied by a drastic activity decrease far before unfolding of the protein is detected by spectroscopy or proteolysis. In addition to ionic effects, a tightening of the protein structure at low guanidine hydrochloride concentrations is suggested to be responsible for ribonuclease A inactivation.     

Effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease

We have investigated the effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. The sensitivity of nonglycosylated RNase A to trypsin and chymotrypsin was compared with three glycosylated species of RNase B which differed with respect to the size of the carbohydrate chain. Two forms of glycosylated RNase B were isolated by concanavalin A-Sepharose affinity chromatography, and each was shown to contain a single carbohydrate chain composed of GlcNAc2Man1 (RNase B") or GlcNAc2Man5-8 (RNase B). A third form (RNase B'), with oligosaccharide composed of GlcNAc2Man4, was prepared by partial digestion of RNase B with alpha-mannosidase. Fully glycosylated RNase B was found to be 6-10 times more resistant to trypsin digestion than nonglycosylated RNase A. RNase B' and B", with intermediate chain sizes, were 3.0- and 1.3-fold more resistant to trypsin digestion than RNase A, respectively. With chymotrypsin, however, differences in rates of digestion were much less marked, with a maximum difference of 3-fold between RNase A and B. In addition, we found that the specificity of the primary trypsin (Arg 33-Asp 34 bond) or chymotrypsin (Tyr 25-Cys 26 bond) cleavage site was not affected by the presence or size of the oligosaccharide chain. These results are consistent with the view that the size of the oligosaccharide chain and its proximity to the primary or rate-limiting cleavage site are important for expression of the carbohydrate protection against proteolytic degradation, which thus appears to be mediated by steric hindrance.     

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 (T_m=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.     

Proton NMR assignments and regular backbone structure of bovine pancreatic ribonuclease A in aqueous solution

Proton NMR assignments have been made for 121 of the 124 residues of bovine pancreatic ribonuclease A (RNase A). During the first stage of assignment, COSY and relayed COSY data were used to identify 40 amino acid spin systems belonging to alanine, valine, threonine, isoleucine, and serine residues. Approximately 60 other NH-alpha CH-beta CH systems were also identified but not assigned to specific amino acid type. NOESY data then were used to connect sequentially neighboring spin systems; approximately 475 of the possible 700 resonances in RNase A were assigned in this way. Our assignments agree with those for 20 residues assigned previously [Hahn, U., & Rüterjans, H. (1985) Eur. J. Biochem. 152, 481-491]. Additional NOESY correlations were used to identify regular backbone structure elements in RNase A, which are very similar to those observed in X-ray crystallographic studies [Wlodawer, A., Borkakoti, N., Moss, D. S., & Howlin, B. (1986) Acta Crystallogr. B42, 379-387].     

Mutations in domain a' of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A

Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a', and two inactive ones, b and b', all four domains having the thioredoxin fold. Domain b' contains the primary peptide binding site, but a' is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Delta455-457, and abb', and the individual domains a and a'. The first two mutants contained alterations in the last alpha helix of domain a', while the third lacked the entire domain a'. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Delta455-457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb', and over 15-fold in the cases of the individual domains a and a'. In addition, PDI F449R and PDI abb' affected the distribution of folding intermediates. Domains a and a' catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process.     

Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.