Helix-destabilization of deoxyribonucleic acid and poly[d(A-T).d(A-T)] by bovine seminal ribonuclease

A ribonuclease isolated earlier from bovine seminal plasma by DNA-affinity chromatography (Ramakrishnamurti, T. and Pandit, M.W. (1983) J. Chromatogr. 260, 216-222) has now been shown by thermal denaturation studies to destabilize the double-helical structure of DNA and poly[d(A-T).d(A-T)]. Thermal denaturation profiles of DNA in the presence of the protein are much more complicated due to the denaturation of protein itself in the temperature range over which DNA predominantly melts. The protein shows relatively stronger affinity towards denatured DNA as compared to native DNA. The action of micrococcal nuclease on DNA and its complexes with ribonuclease A and bovine seminal ribonuclease indicates that both of these proteins destabilize the double-helical structure of native DNA and thereby render the DNA more sensitive to the micrococcal nuclease.     

Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridges.

The hypothesis previously advanced that interchain disulfide bridges link the two identical subunits of bovine seminal ribonuclease BS-1 has been confirmed. The sedimentation rate and the electrophoretic mobility of the protein are not affected by denaturing agents unless thiol reagents are present in the denaturation mixtures. Reduction under controlled conditions results in the immediate cleavage of only 2 disulfide bonds out of 10 percent in the dimeric protein. Under these conditions, and the results do not change when partial reduction is followed by S-alkylation, 30% of the protein dissociates, while the remaining is found to consist of a dimeric species easily dissociable by denaturing agents without addition of thiol reagents. This indicates that the dimeric structure of seminal ribonuclease is maintained not only by disulfide bridges, but also by noncovalent forces. The protein derivative prepared by selective reduction and alkylation has been identified as monomeric bis-S-carboxymethylcysteine-31,32-ribonuclease BS-1. This is on the basis of the characterization of the 14C-labeled S-carboxymethylated peptides isolated from a thermolytic hydrolysate of the derivative prepared with iodo-2-[14C]acetic acid. Monomeric, selectively alkylated ribonuclease BS-1 is stable and catalytically active. The importance of such a derivative is discussed both in the light of the recent studies on the biological actions of seminal ribonuclease and as the fourth component of an experimental system of ribonucleases consisting of two homologous dimers (bovine seminal ribonuclease BS-1 and dimerized bovine pancreatic ribonuclease A) and two homologous monomers (ribonuclease A and the monomeric derivative of ribonuclease BS-1.     

Reacquisition of quaternary structure by fully reduced and denatured seminal ribonuclease

Air-regenerated monomers of bovine seminal ribonuclease have been found capable of reassociating into native dimers, whereas monomers refolded in the presence of a glutathione redox mixture do not reassociate into dimers [Smith, K. G., D'Alessio, G. and Schaffer, S. W. (1978) Biochemistry 17, 2633-2638]. The crucial step in the process of regeneration of dimers is an isomerization step, which the newly refolded monomers undergo in order to reassociate into dimers. The two sulfhydryls at sequence positions 31 and 32 of the seminal RNAase chain, forming in the native dimer the intersubunit disulfides, have been found to have an important role in the refolding of the monomeric intermediates, as well as in the regeneration of dimers.     

Intrachain disulfide bridges of bovine seminal ribonuclease

The pairing of the four intrachain disulfide bonds of bovine seminal ribonuclease, a dimeric protein isolated from bovine seminal plasma, has been established by the isolation and characterization of the cystine peptides obtained from a thermolytic-tryptic hydrolysate of the protein. These disulfide bonds involve eight half-cystine residues located in the protein subunit chain at sequence positions identical with those of the eight half-cystine residues of the strictly homologous chain of bovine pancreatic ribonuclease. The results reported show that these eight 'homologous' half-cystine residues pair in seminal ribonuclease exactly as they do in pancreatic ribonuclease. They also indirectly confirm that the remaining two half-cystine residues present in each chain of the seminal enzyme are involved in intersubunit bonds.     

A calorimetric approach to the study of the interactions of cytidine-3'-phosphate with bovine seminal ribonuclease

A calorimetric study at 25 degrees C is reported on the interaction between allosteric bovine seminal ribonuclease and cytidine-3'-phosphate. The results are compared with those obtained under identical experimental conditions for the interaction of pancreatic ribonuclease A and the same nucleotide. The analysis of the data provides evidence that the binding sites of seminal ribonuclease for cytidine-3'-phosphate are not equivalent, in agreement with previous equilibrium dialysis studies. A model with two sites with different affinities toward the nucleotide, the site with higher affinity resembling the binding site of pancreatic ribonuclease, is proposed. The values calculated for the thermodynamic parameters provide an insight of the forces involved in the interaction of the two enzymes with the nucleotide.     

Selective reduction of seminal ribonuclease by glutathione

Incubation of seminal ribonuclease with glutathione leads to the formation of a monomeric species which exhibits twice the specific activity of the native dimer. The monomer was found to possess two mixed disulfides of glutathione at residues 31 and 32, the residues ordinarily involved in the intermolecular disulfide bonds linking the subunits of the native dimer. Formation of the monomer results in only minor changes in the far ultraviolet circular dichroism spectra. The rate of the glutathione-facilitated dissociation reaction is fairly slow, requiring 60 min for completion. Attempts to dimerize the monomer all failed, implying that the dissociation reaction is irreversible. The glutathione reduced monomer was compared with the monomer formed during the regeneration of reduced, denatured bovine seminal ribonuclease in the presence of glutathione. By all criteria examined, the two monomeric forms are identical. It is concluded that the mixed disulfide monomer is the favored form of the enzyme in the presence of glutathione.     

Crystal structure of the dimeric unswapped form of bovine seminal ribonuclease

Bovine seminal ribonuclease is a unique case of protein dimorphism, since it exists in two dimeric forms, with different biological and kinetic behavior, which interconvert into one another through three-dimensional swapping. Here we report the crystal structure, at 2.2 A resolution, of the unswapped form of bovine seminal ribonuclease. Besides completing the structural definition of bovine seminal ribonuclease conformational dimorphism, this study provides the structural basis to explain the dependence of the enzyme cooperative effects on its swapping state.     

The ribbon of hydrogen bonds and the pseudomolecule in the three-dimensional structure of globular proteins. III. Bovine pancreatic ribonuclease A and bovine seminal ribonuclease

The model of the three-dimensional structure of globular proteins, which is based on a ribbon of hydrogen bonds along the whole of the backbone, is now applied to the comparison between monomeric bovine pancreatic ribonuclease A and dimeric bovine seminal ribonuclease. Some waters are involved in the hydrogen bonding of the ribbon, and the protein molecule plus these waters forms a pseudomolecule. The conformations of the three backbones are essentially identical and the three ribbons of hydrogen bonds are conserved with greater than 90% accuracy. We suggest that the conservation of the backbone conformations of the two molecules is a consequence of the conservation of the ribbons of hydrogen bonds. There are 16 simple mutations between the two molecules, of which 15 involve only side-chain groups with no more than one hydrogen bond to the backbone. Such mutations are not sufficient to change the ribbon of hydrogen bonds and hence there is no change in the backbone conformation. Generalizing this result, we suggest that the conservation of the ribbon is the reason why single point mutations rarely change the conformation of the backbone of the globular proteins.     

Evolution of oligomeric proteins. The unusual case of a dimeric ribonuclease.

The model system made up of a monomeric and a dimeric ribonuclease of the pancreatic-type superfamily has recently attracted the attention of investigators interested in the evolution of oligomeric proteins. In this system, bovine pancreatic ribonuclease (RNase A) is the monomeric prototype, and bovine seminal ribonuclease (BS-RNase) is the dimeric counterpart. However, this evolutionary case is unusual, as BS-RNase is the only dimeric member of the whole large superfamily comprising more than 100 identified members from amphibia, aves, reptilia and mammalia. Furthermore, although the seminal-type RNase gene can be traced back to the divergence of the ruminants, it is expressed only in a single species (Bos taurus). These unusual findings are discussed, as well as previous hypotheses on the evolution of seminal RNase. Furthermore, a new 'minimalist' hypothesis is proposed, in line with basic principles of structural biology and molecular evolution.     

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.     

Sequences related to the ox pancreatic ribonuclease coding region in the genomic DNA of mammalian species

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants.In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species.We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.Correspondence to: A. Furia     

The major basic proteins of bull seminal vesicle secretion

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.     

The role of the hinge loop in domain swapping. The special case of bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16-22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase.     

Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation

We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.     

Fourth derivative UV-spectroscopy of proteins under high pressure II. Application to reversible structural changes

The structural basis and the thermodynamics of pressure induced reversible spectral transitions in the fourth derivative ultraviolet absorbance spectra of proteins were analysed as described in the preceding paper. Three proteins were studied: adrenodoxin (a small iron-sulphur protein that serves as an electron donor for cytochrome P450scc), ribonuclease A, and methanol dehydrogenase (a tetrameric protein). Fourth derivative spectroscopy is used to probe important mechanistic aspects of these proteins. For adrenodoxin, the results suggest that one or two phenylalanines interact with the iron-sulphur redox centre. High pressure denaturation of ribonuclease leads to a molten globule like structure that also occurs as an intermediate in the high temperature induced denaturation process. This state is characterised by the local dielectric constant in the vicinity of tyrosines. Methanol dehydrogenase was found to be very stable towards pressure. High pressure appears to strengthen the interaction between the two -subunits possibly through the increased interaction of four tryptophans with other aromatic amino acids.     

The swapping of terminal arms in ribonucleases: comparison of the solution structure of monomeric bovine seminal and pancreatic ribonucleases

Bovine seminal ribonuclease (BS-RNase), the only dimeric protein among the pancreatic-like ribonucleases, is endowed with special structural features and with biological functions beyond enzymatic activity. In solution, the protein exists as an equilibrium mixture of two forms, with or without exchange (or swapping) of the N-terminal arms. After selective reduction and alkylation of the two intrachain disulfide bridges, the dimeric protein can be transformed into a monomeric derivative that has a ribonuclease activity higher than that of the parent dimeric protein but is devoid of the special biological functions. A detailed investigation of the structural features of this protein in solution, in comparison with those of other monomeric ribonucleases, may help unveil the structural details which induce swapping of the N-terminal arms of BS-RNase. The solution structure of the recombinant monomeric form of BS-RNase, as determined by 3D heteronuclear NMR, shows close similarity with that of bovine pancreatic ribonuclease (RNase A) in all regions characterized by regular elements of secondary structure. However, significant differences are present in the flexible regions, which could account for the different behavior of the two proteins. To characterize in detail these regions, we have measured H/D exchange rate constants, temperature coefficients and heteronuclear NOEs of backbone amides for both RNase A and monomeric BS-RNase. The results indicate a large difference in the backbone flexibility of the hinge peptide segment 16-22 of the two proteins, which could provide the molecular basis to explain the ability of BS-RNase subunits to swap their N-terminal arms.     

Topological comparison of two helix destabilizing proteins: ribonuclease A and the gene 5 DNA binding protein

A topological comparison of the two helix destabilizing proteins, pancreatic ribonuclease A and the gene 5 DNA binding protein of bacteriophage fd has been completed utilizing the available high resolution tertiary structures of each protein. The results indicate these two proteins are structurally if not also evolutionarily related. Regions of closet topological equivalence occur between beta loops directly involved in nucleotide binding or are required for the maintenance of their respective oligonucleotide binding channels. In addition, there is a similar placement of critical amino acid side chains about the binding site. Further evidence for this structural relationship is obtained by comparison of structural data for the mode of complexation of polynucleotides to each protein. The results of topological comparison suggest the essential property shared by helix destabilizing proteins, whether specialized DNA binding proteins such as G5BP or proteins with other primary functional roles, like ribonuclease A, is the presence of an elongated oligonucleotide binding channel. Although ribonuclease A and G5BP are structurally related, it seems likely any protein with this structural feature will exhibit a helix destabilizing capacity. This conclusion is supported by the diversity of molecular characteristics shown by other proteins having this activity.     

Impact of an easily reducible disulfide bond on the oxidative folding rate of multi-disulfide-containing proteins.

The burial of native disulfide bonds, formed within stable structure in the regeneration of multi-disulfide-containing proteins from their fully reduced states, is a key step in the folding process, as the burial greatly accelerates the oxidative folding rate of the protein by sequestering the native disulfide bonds from thiol-disulfide exchange reactions. Nevertheless, several proteins retain solvent-exposed disulfide bonds in their native structures. Here, we have examined the impact of an easily reducible native disulfide bond on the oxidative folding rate of a protein. Our studies reveal that the susceptibility of the (40-95) disulfide bond of Y92G bovine pancreatic ribonuclease A (RNase A) to reduction results in a reduced rate of oxidative regeneration, compared with wild-type RNase A. In the native state of RNase A, Tyr 92 lies atop its (40-95) disulfide bond, effectively shielding this bond from the reducing agent, thereby promoting protein oxidative regeneration. Our work sheds light on the unique contribution of a local structural element in promoting the oxidative folding of a multi-disulfide-containing protein.     

Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease

Binding to cell membrane, followed by translocation into the cytosol and RNA degradation, is a necessary requirement to convert a ribonuclease into a cytotoxin for malignant tumor cells. In this paper, we investigate the membrane binding attitude of bovine seminal ribonuclease (BS-RNase) and its variant G38K-BS-RNase, bearing an enforced cluster of positive charges at the N-termini surface. By using a combination of biophysical techniques, including CD, SPR and ESR, we find for the two proteins a common, two-step mechanism of interaction with synthetic liposomes, an initial binding to the bilayer surface, driven by electrostatic interactions, followed by a shallow penetration in the lipid core. Protein binding effectively perturbs lipid packing and dynamics. Remarkably, the higher G38K-BS-RNase membrane interacting capability well correlates with its increased cytotoxicity for tumor cells. Overall, these studies shed light on the mechanism of membrane binding and perturbation, proving definitely the importance of electrostatic interactions in the cytotoxic activity of BS-RNase, and provide a rational basis to design proteins with anticancer potential.