Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 70-residue analog of RNase S-protein was synthesized by the solid phase method. It was obtained by omitting the NH2 terminus from positions 21 to 25 and the segments 36 to 40, 58 to 73, 87 to 96, and 113 to 114. Four residues were inserted to link the ends formed by the deletions. Half-cystine residues that had not been part of the deletions were replaced by alanine or leucine residues. The synthetic polypeptide was separated by gel filtration into a dimer and a monomer. Both fractions were purified further by ion exchange chromatography. The dimeric 70-residue S-protein analog had a specific activity of approximately 4% using RNA as substrate. It also cleaved other substrates of RNase A such as 5'-(3'-cytidylyl)-guanosine, 5'-(3'-uridylyl)-guanosine, and polycytidylic acid. The monomer of the 70-residue analog was less active but showed the same substrate specificity as the dimer. It was found that both fractions of the synthetic S-protein analog catalyzed only the transphosphorylation step of the RNase A mechanism and had very little if any activity in the hydrolysis step. Addition of natural S-peptide or S-protein did not increase the activity in the transphosphorylation reaction but greatly enhanced the reaction rate of the hydrolysis step. IN THE PRESENCE OF S-peptide, both monomeric and dimeric 70-residue S-protein, both monomeric and dimeric 70- residue S-protein analog had approximately 8% activity using cyclic cytidine 2':3'-monophosphate as substrate. The mixtures of monomer and dimer of the synthetic S-protein analog with natural S-protein generated even higher activities (151 and 74%, respectively) against this substrate despite the fact that the NH2-terminal portion of the natural enzyme (including His 12) was missing in both components of the two complexes. The 70-residue S-protein analog was completely inactive against DNA and (with one exception) against substrates for RNase T1. The close agreement of the substrate specificity of the synthetic analog with that of native RNase A in the transphosphorylation step suggested a remarkable conservation of the configuration of the active site despite drastic changes of the primary structure of the parent molecule. Possible implications of these results for the mechanism of action of RNase A are discussed.
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PMID:A synthetic 70-amino acid residue analog of ribonuclease S-protein with enzymic activity. 111 95

The relationship of structure to function in the recognition of ribonuclease S-peptide by S-protein was studied by several methods. Liquid phase peptide synthesis was employed to generate analogs of S-peptide in which from 1 to 8 residues were deleted from the NH2-terminal end of the S-peptide. Additional derivatives were made by substitutions in the NH2-terminal three amino acids or by modifying the S-peptide analogs by trifluoroacetylation. The analogs were generated in the following way. S-Peptide was cleaved with chymotrypsin. The fragment obtained, RNase(9-20), was purified and lengthened step by step using liquid phase peptide synthesis. A second set of analogs were prepared by cleavage of CF3CO-S-peptide with elastase and the resulting CF3CO-RNase(7-20), similarly lengthened. The various analogs of S-peptide were tested in their capacity to combine with S-protein and regenerate biological activity as measured by Vmax and Kb. This work shows a positive contribution of every one of the first 8 NH2-terminal residues of S-peptide to the molecular recognition of S-protein in the presence of RNA substrate. Substitution of the first 3 residues by alanine or blocking of the free amino groups decreases recognition, indicating that the original primary structure is the most favorable one.
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PMID:Ribonuclease S-peptide. A model for molecular recognition. 125 70

A defective S-allele, S(o), and a functional S-allele, Sx, have previously been found to be retained in an F1 hybrid of a self-compatible commercial cultivar of Petunia hybrida. Pistil proteins associated with these two alleles have also been identified. Their amino-terminal sequences have been found to share a high degree of similarity with those of S-proteins characterized from self-incompatible solanaceous species. Here we report the isolation and sequencing of cDNAs encoding S(o)- and Sx-proteins. Their deduced amino acid sequences contain all the consensus primary structural features of S-proteins from self-incompatible solanaceous species. Both proteins also have ribonuclease activity. The implications of these findings are discussed in relation to the presumed function of the S-protein in the self-incompatibility interaction.
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PMID:Cloning and sequencing of cDNAs encoding two S proteins of a self-compatible cultivar of Petunia hybrida. 134 83

Hydrophobic interactions between the S-peptide and S-protein in the ribonuclease-S complex are probed using molecular dynamics simulations and free energy calculations. Three successive mutations at the buried position Met13 are simulated: Met----Leu, Leu----Ile, and Ile----Val, for which X-ray structures and experimental thermodynamic data are available. The calculations give theoretical estimates of the changes in binding free energies associated with these mutations. The calculated free energy differences are small (0-1.6 kcal/mol), in agreement with experiment. However the simulated structures deviate significantly from the experimental ones (mean deviation approximately 1.5-2 A), and a large uncertainty in the calculated free energies (1-2 kcal/mol) arises from the multiple minimum problem. Indeed, multiple conformations are available to the side chains around the mutation site, and the sampling of dihedral rotamer transitions is limited, despite long simulations. Fluctuations within each local minimum give rise to a small statistical error. However the uncertainty due to multiple conformations is much greater than the uncertainty due to random statistical errors. In our work, an artificial cancellation of errors arose because we studied conformations of the RNase complex and of the S-peptide that were very similar. In general, the criterion for a precise simulation is not merely to reduce the random statistical error, as has been suggested, but rather to sample all the important local minima along the mutation pathway, and to reduce the statistical error for each one. Our calculations suggest that the packing changes associated with the mutations are energetically small and localized, and largely cancel when the complex and the S-peptide are compared. Solvation of the methionine side chain partial charges in the S-peptide and the complex appear to be energetically equivalent, so that removing them (as in Met13----Leu, Ile, Val) does not affect binding. Enthalpy and entropy changes could not be estimated reliably.
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PMID:Thermodynamics of protein-peptide interactions in the ribonuclease-S system studied by molecular dynamics and free energy calculations. 139 Jun 51

Ribonuclease S (RNase-S) is a complex that consists of two proteolytic fragments of bovine pancreatic ribonuclease A (RNase-A): the S-peptide (residues 1-20) and S-protein (residues 21-124). We have refined the crystal structures of three RNase-S complexes. The first two contain the full-length 20-residue S-peptide and were studied at pHs of 4.75 and 5.5. The third one consists of a truncated form of S-peptide (residues 1-15) and was studied at pH 4.75 as the reference structure for a series of mutant peptide complexes to be reported separately. Excluding residues 16-23 which are either missing (in the S15 complex) or disordered (in both S20 complexes), all three structures refined at 1.6-A resolution are identical within the estimated errors in the coordinates (0.048 A for the backbone atoms). The R-values, residual error, range from 17.4% to 18.6%. The final model of S20, pH 4.75, includes 1 sulfate and 84 water molecules. The side chains of 11 residues were modeled in two discrete conformations. The final structures were independent of the particular RNase-A or RNase-S used as a starting model. An extensive comparison with refined crystal structures of RNase-A reveals that the core of the molecule which is held together with extensive hydrogen bonds is in identical pattern in all cases. However, the loop regions vary from one structure to another and are often characterized by high B-factors. The pattern of thermal parameters appears to be dependent on crystal packing and correlates well with the accessibility calculated in the crystal. Gln60 is a conserved residue in all sequences known to date for this class of ribonucleases. However, it is the only residue that is clearly defined in an unfavorable position (phi = -100 degrees, psi = -130 degrees) on the Ramachandran plot. The origin of the substantial differences between RNase-A and RNase-S in stability to both acid and temperature denaturation and in susceptibility to proteolysis at neutral pH is not obvious in our visual comparison of these two structures.
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PMID:Refinement of the crystal structure of ribonuclease S. Comparison with and between the various ribonuclease A structures. 146 19

Seven hydrophobic residues ranging in size from glycine to phenylalanine have been substituted for the wild-type methionine residue at position 13 in a 15-residue truncated version (S15) of S-peptide, the small component of ribonuclease S. Complexes of both S-15 and the seven variants with S-protein yielded isomorphous crystals. The structures of all eight complexes have been refined to final R-factors in the range of 17-19%. [See Kim, E. E. Varadarajan, R., Wyckoff, H. W., and Richards, F. M. (1992) Biochemistry (preceding paper in this issue) for the description of the reference S-15 complex.] Multiple side-chain conformations were seen for six residues in all of the complexes and for two to three additional residues in at least some of the complexes. Three of the complexes, Gly, Ala, and alpha-amino-n-butyric acid (ANB), contained a single water molecule in the cavity near residue 13 that makes three hydrogen bonds to protein atoms. Although space is available, no evidence for additional water in this region, ordered or disordered, was found. The atoms in the cavity wall tend to shrink the cavity by moving in on the small residues and to swell the cavity by moving out for the larger Phe substitution. A swelling seen with leucine was attributed to a shape effect since Leu, Ile, and Met all have the same volume. A slight volume contraction of the collection of interior residues outside of the region of position 13 was also noted. (All changes noted are in the direction to maintain a constant packing density averaged over the whole protein.) Leu51, a surface hydrophobic residue, moved considerably in the G, A, and ANB complexes in directionswhich would tend to decrease the cavity volume. The only other major change in position, 1.5 A, was the 66-69 loop, which is about 25 A from position 13. His12, Phe120, and Asp121 appear to be involved in this movement, but the connection with position 13 is not clear at all. The thermodynamic data on the association reaction for all of these complexes have been previously reported [Connelly, P. R., Varadarajan, R., Sturtevant, J. M., & Richards, F. M. (1990) Biochemistry 29, 6108-6114; Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426]. Some comments are offered on our initial attempts to correlate the structural changes with the changes in the thermodynamic parameters.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Crystallographic structures of ribonuclease S variants with nonpolar substitution at position 13: packing and cavities. 146 20

Glucocorticoid treatment induces programmed cell death (apoptosis) in susceptible T lymphocytes. We have previously isolated and characterized 13 genes induced by agents that cause apoptosis in the murine thymoma cell line, WEHI-7TG. One of these genes, now designated Tcl-30, encodes a 2.4-kb mRNA that is specifically expressed in thymus. Sequence analysis of a full-length cDNA for Tcl-30 reveals a potential open reading frame of 454 amino acids that shares sequence identity to a human placental-specific protein of unknown function, PP11. The putative protein encoded by Tcl-30 also contains a cysteine-rich somatomedin B-like domain found in vitronectin, PP11, and the plasma cell membrane glycoprotein, PC-1. Subpopulations of murine thymocytes sorted on the basis of their expression of the CD4, CD8, and surface heat-stable Ag (HSA) were analyzed by an RNase protection assay to determine the expression of Tcl-30 as a function of T cell development. Tcl-30 was expressed exclusively in the HSA+ T cell populations (CD4-CD8-HSA+; CD4+CD8-HSA+; CD4-CD8+HSA+; and CD4+CD8+HSA+) and not in the HSA- single positive T cell populations (CD4+CD8- or CD4-CD8+) of the thymus or spleen. Therefore, we conclude that Tcl-30 expression is lost during T cell maturation and is absent at the most mature stages of T cell development. The function of Tcl-30 is unknown; however, the CD4+CD8+ double-positive subpopulation expressing Tcl-30 represents thymocytes destined to undergo massive intrathymic cell death. The possibility that Tcl-30 expression may define a population of T lymphocytes that is sensitive to glucocorticoid-mediated apoptosis is discussed.
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PMID:Tcl-30, a new T cell-specific gene expressed in immature glucocorticoid-sensitive thymocytes. 150 80

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.
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PMID:Heat capacity changes for protein-peptide interactions in the ribonuclease S system. 173 99

Ribonuclease inhibitor (RI) is a protein that forms a very tight complex with ribonucleases (RNases) of the pancreatic type. RI contains 30 thiol groups, some of which are important for the enzyme-inhibitor interaction. To examine which thiols are affected by the binding of RNase, differential labeling experiments were performed. Reaction of porcine RI with the cysteine-specific labeling reagent 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid resulted in labeling of an average of 7.4 of the 30 cysteinyl residues. Binding of bovine pancreatic RNase A caused a 3.2-fold reduction in the extent of modification. Peptide mapping showed that in free RI, Cys-57, -371, and -404 were labeled to the greatest extent (yield, 0.4-0.6 mol/mol). RNase A did not protect Cys-57 against modification, whereas the labeling of Cys-371 and -404 was reduced by more than 90%. A second group of residues was labeled to a lesser extent in free RI (yield, 0.04-0.2 mol/mol). Within this group 11 residues were protected by RNase A by more than 90%, 2 were not affected at all, and 7 were protected between 10 and 90%. Seven cysteinyl residues in RI that were protected in the RI.RNase A complex were no longer protected in the RI.S-protein complex. These residues were mainly present in the N-terminal region of RI. However, when the S-peptide was included to yield the RI.RNase S complex, the same pattern of labeling was obtained as with the RI.RNase A complex. Addition of the S-peptide alone had no effect on the labeling. The implications of these observations with respect to RNase binding areas of RI are discussed in relation to the results obtained from the analysis of active RI molecules that contain deletions.
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PMID:Studies on the interaction of ribonuclease inhibitor with pancreatic ribonuclease involving differential labeling of cysteinyl residues. 174 89

We have isolated and sequenced cDNAs for S2- and S3-alleles of the self-incompatibility locus (S-locus) in Solanum chacoense Bitt., a wild potato species displaying gametophytic self-incompatibility. The S2- and S3-alleles encode pistil-specific proteins of 30 kDa and 31 kDa, respectively, which were previously identified based on cosegregation with their respective alleles in genetic crosses. The amino acid sequence homology between the S2- and S3-proteins is 41.5%. This high degree of sequence variability between alleles is a distinctive feature of the S-gene system. Of the 31 amino acid residues which were previously found to be conserved among three Nicotiana alata S-proteins (S2, S3, and S6) and two fungal ribonucleases (RNase T2 and RNase Rh), 27 are also conserved in the S2- and S3-proteins of S. chacoense. These residues include two histidines implicated in the active site of the RNase T2, six cysteines, four of which form disulfide bonds in RNase T2, and hydrophobic residues which might form the core structure of the protein. The finding that these residues are conserved among S-proteins with very divergent sequences suggests a functional role for the ribonuclease activity of the S-protein in gametophytic self-incompatibility.
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PMID:Cloning and sequencing of cDNAs encoding two self-incompatibility associated proteins in Solanum chacoense. 226 40


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