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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Four, widely used, ribonucleases were found to protect their substrates from acid precipitation by causing, evidently, a modification of their physicochemical properties. The protection was dependent on the kind of substrate while the ratio of protective to nucleolytic activity varied widely between the four enzymes. The protection was enhanced by some nucleotides like UMP, CMP and IMP and decreased in the presence of several bivalent ions like Zn++, Co++ and Cu++. It was completely abolished when the substrates were hybridized with their complementary ribohomopolymers. In the case of bovine pancreatic ribonuclease, the part of the molecule which was responsible for the protective activity was localized on the enzyme domain characterized as S-protein, which lacks nucleolytic activity. The observed property of ribonucleases could lead to false data when the measurement of TCA-soluble material is the method used to follow the purification of ribonucleases or to study their activity. It was also found that ribonuclease S-protein enhances the catalytic activity of B. Cereus RNAse. S-protein could potentiate other RNAses activity like onconase, which has recently been used as an anticancer agent.
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PMID:Ribonucleases protect RNA from acid precipitation. 948 43

Peptides deduced from peptide libraries may serve as affinity ligands for protein purification. Identification of a ligand that binds the protein of interest depends highly on the screening method used. One approach which offers simple and direct detection involves screening a solid-phase peptide library against a radiolabeled target protein. We have developed a radiological screening method, using 14C as a radioactive label, that offers high resolution and sensitivity. Less than 100 DPM/bead are detectable after a one-day exposure using autoradiography. The validity of the technique was illustrated by screening a solid-phase hexameric-peptide library spiked with YNFEVL-beads against 14C-labeled ribonuclease S-protein. For this particular system, the amount of protein bound to a single bead was estimated to be in the femtomolar range with a peptide:protein ratio of 500:1. Finally, a portion of the library was screened against 14C-labeled fibrinogen. Three peptides deduced from the library, WQEHYN, WQETYQ, and YENYGY, purified fibrinogen from a mixture with albumin.
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PMID:Screening of combinatorial peptide libraries: identification of ligands for affinity purification of proteins using a radiological approach. 992 97

We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the alpha-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the alpha-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of -0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state.
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PMID:A specific transition state for S-peptide combining with folded S-protein and then refolding. 1005 87

A theoretical model is presented to study the stepwise thermal unfolding of globular proteins using the stabilizing/destabilizing characters of amino acid residues in protein crystals. A multiple regression relation connecting the melting temperature and the amounts of stabilizing and destabilizing groups of residues in a protein, when used for the thermal behavior of peptide segments, provides reliable results on the stepwise unfolding nature of the protein. In ribonuclease A, the shell residues 16-22 are predicted to unfold earlier in the temperature range 30-45 degrees C; the beta-sheet structures undergo thermal denaturation as a single cooperative unit and there is evidence indicating the segment 106-118 as a nucleation site. In ribonuclease S, the S-peptide unfolds earlier than S-protein. The predicted average and the range of melting temperatures, and the folding pathways of a set of globular proteins, agree very well with the experimental results. The results obtained in the present study indicate that (i) most of the nucleation parts possess high relative thermal stability, (ii) the unfolded state retains some residual structure, and (iii) some segments undergo gradual and overlapping thermal denaturation.
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PMID:On the thermal unfolding character of globular proteins. 1088 67

The 15-meric S-tag is a truncated form of the S-peptide, which builds together with the 103 amino acid large S-protein the whole ribonuclease S-protein. Its small size and excessive solubility have made the S-tag an excellent fusion partner in the production of recombinant proteins, and a large variety of applications have been reported using the S-tag as a carrier. While S-tagged proteins were mostly detected and analyzed so far by use of their affinity to S-proteins, monoclonal antibodies (MAbs) for this tag have been not available. The generation of antibodies specific for S-tagged proteins is expected to broaden the range of applications of such S-tag fused recombinant proteins, and in this context, a novel MAb termed ATOM-2 was generated that specifically binds S-tagged proteins, which have been expressed using pET-vectors. Antigen specificity of ATOM-2 was confirmed in Western blot and enzyme-linked immunoadsorbent assay analysis, and using a series of amino acid deletion mutants, the binding epitope of ATOM-2 was precisely mapped. This showed that ATOM-2 recognizes the C-terminal part of the 15-meric S-tag in context with a few residues of vector encoded sequences. The core sequence for ATOM-2 binding epitope is "His-Met-Asp-Ser-Pro-Asp-Leu-Gly-Thr," which is present in all pET-expression vectors encoding S-tag fusion proteins. Because the ATOM-2 binding region does not overlap with the S-protein binding sequence, a convenient tool is provided for the simultaneous or alternative detection, purification, and analysis of recombinant S-tagged proteins to conventional S-proteins.
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PMID:Detection of pET-vector encoded, recombinant S-tagged proteins using the monoclonal antibody ATOM-2. 1128 23

We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation.
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PMID:S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis. 1264 45

The interaction between ribonuclease (RNase) S-protein and S-peptide is examined by studying their limited oxidation within the RNase-S complex and free forms using radicals. The limited oxidation of the RNase-S complex and each component is effected through their reaction with a high flux of oxygen-based radicals generated by an electrical discharge within an electrospray ion source. Their exposure to radicals occurs on short millisecond time scales and has been consistently found not to cause any measurable structural damage or conformational change to proteins in a number of published reports. Consistent with these studies, S-peptide is preferentially protected from reactions with radicals under conditions in which it is bound to S-protein. Conversely, a region of S-protein comprising residues 96-100 constitutes the S-peptide binding domain based on its diminished reactivity with radicals within the RNase-S complex over the free S-protein. The results, for the first time, demonstrate the use of radicals generated by an electrical discharge to study protein complexes.
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PMID:Study of the ribonuclease-S-protein-peptide complex using a radical probe and electrospray ionization mass spectrometry. 1270 85

Specific peptidyl linkers that result in the heterodimerization of functional proteins, which is catalyzed by microbial transglutaminase from Streptomyces mobaraensis (MTG), were generated based on a ribonuclease S-peptide using site-directed mutagenesis. The peptidyl linkers designated as Lys-tag and Gln-tag were designed to possess sole reactive Lys or Gln residue that was amenable for selective Lys-Gln cross-linkage of different proteins. Green fluorescent protein variants, ECFP and EYFP, were employed as model proteins, and those Lys- and Gln-tags were fused to the N-termini of ECFP and EYFP, respectively. As a result, we succeeded in solely obtaining the ECFP-EYFP heterodimer without forming multiply cross-linked byproducts. It was found that the reactivity of peptidyl linkers varied according to the type of amino acid to be replaced. Peptidyl linkers with a basic amino acid (Arg) exhibited the highest reactivity in the cross-linking reaction, suggesting the cationic residue substrate preference of MTG. Kinetic analysis utilizing fluorescent resonance energy transfer (FRET), that is only observed upon the heterodimeric ECFP-EYFP conjugation, revealed that the amino acid replacement contributed to the acceleration of cross-linking reactions by increasing catalytic turnover (k(cat)), rather than substrate binding affinity (K(m)). Finally, using a ribonuclease S-protein, the manipulation of enzymatic protein cross-linking based on specific S-peptide:S-protein interactions was explored. Since newly designed Lys- and Gln-tags retained binding affinities to the S-protein, the heterodimerization was perfectly restrained by wrapping them with the S-protein. The results suggest the possibility of limited protein conjugation by tuning steric hindrance against the MTG. Tailoring enzymatic posttranslational modifications with either engineering peptidyl substrates or by taking specific peptide-protein interactions into consideration may facilitate the development of a new sequential protein conjugation method for the preparation of multifunctional protein.
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PMID:Peptidyl linkers for protein heterodimerization catalyzed by microbial transglutaminase. 1514 76

A new mass spectrometry identifiable cross-linking strategy has been developed to study protein-protein interactions. The new cross-linker was designed to have two low-energy MS/MS-cleavable bonds in the spacer chain to provide three primary benefits: First, a reporter tag can be released from cross-link due to cleavage of the two labile bonds in the spacer chain. Second, a relatively simple MS/MS spectrum can be generated owing to favorable cleavage of labile bonds. And finally, the cross-linked peptide chains are dissociated from each other, and each then can be fragmented separately to get sequence information. Therefore, this novel type of cross-linker was named protein interaction reporter (PIR). To this end, two RINK groups were utilized to make our first-generation cross-linker using solid-phase peptide synthesis chemistry. The RINK group contains a bond more labile than peptide bonds during low-energy activation. The new cross-linker was applied to cross-link ribonuclease S (RNase S), a noncovalent complex of S-peptide and S-protein. The results demonstrated that the new cross-linker effectively reacted with RNase S to generate various types of cross-linked products. More importantly, the cross-linked peptides successfully released reporter ions during selective MS/MS conditions, and the dissociated peptide chains remained intact during MS(2), thus enabling MS(3) to be performed subsequently. In addition, dead-end, intra-, and inter-cross-linked peptides can be distinguished by analyzing MS/MS spectra.
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PMID:Mass spectrometry identifiable cross-linking strategy for studying protein-protein interactions. 1562 10

We label ribonuclease S with a 3 nm Au nanoparticle (NP) by utilizing its two-piece structure. One portion, S-peptide, is mutated with a unique NP attachment site. NP-peptide self-assembles with the other portion, S-protein, to form an active enzyme. NP mobility decreases with peptide labeling and S-protein association. Surface plasmon shifts support conjugation. Higher S-peptide coverages on the NP surface reduce nonspecific adsorption, while sterically hindering assembly of RNaseS. Thiols displace nonspecific adsorption, maximizing site-specific labeling.
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PMID:Labeling ribonuclease S with a 3 nm Au nanoparticle by two-step assembly. 1575 6


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