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:3.1.27.3 (RNase T1)
1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonuclease T1 was crystallized under various conditions. Form I crystals were produced by microdialysis against 53% (v/v) 2-methyl-2,4-pentanediol in 0.01 M sodium acetate, 0.05% 2'-guanylic acid (2'GMP) and 0.02% NaN3 (pH 6.2-7.2). These crystals are tetragonal, space group P41212 and contain two molecules per asymmetric unit; cell dimensions are a = b = 5.86 nm, c = 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01-0.05 M sodium acetate, 0.25-0.5% 2'GMP, 0.02% NaN3 and 2-5 mM calcium acetate (pH 4.0-4.4) in the presence of 50-75% (v/v) 2-methyl-2,4-pentanediol. These crystals are orthorhombic, space group P212121, and contain one molecule per asymmetric unit; cell dimensions are a = 4.66 nm, b = 5.02 nm, c = 4.04 nm (form I) and alpha = 4.44 nm, b = 5.00 nm, c = 4.03 nm (form II). Using high-performance liquid chromatography, it could be shown for all crystal forms that 2'-GMP is bound in the crystals. The molecular ratio between RNase T1 and 2'GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme-nucleotide complex. Heavy-atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three-dimensional X-ray analysis of the RNase-T1 x 2'GMP complex is under way.
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PMID:Crystallization of a complex between ribonuclease T1 and 2'-guanylic acid. 625 Aug 34

Ribonuclease T1 is highly specific for the guanylic acid residue in polyribonucleotides. To clarify the origin of the substrate specificity, the interaction sites of guanylic acid with ribonuclease T1 were investigated by the use of 15N-NMR. 95% 15N-enriched guanosine-3'-phosphate was prepared and mixed with purified ribonuclease T1. 15N-NMR spectra of the mixtures at different concentrations were obtained and compared with that of the 15N-enriched substrate alone. Upon complex formation, a 15N signal assigned to the amino group nitrogen at position 2 of guanine shifted and was significantly broadened, suggesting a strong interaction with the enzyme through the amino group. This observation is consistent with the results of studies on the substrate specificity of chemical modification. Nuclear Overhauser effects of signals assigned to N-7 and N-3 were also changed, but not shift was observed. The observations do not support the occurrence of protonation at N-7 upon complex formation, which was previously proposed.
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PMID:A 15N-NMR study on ribonuclease T1-guanylic acid complex. 627 92

The primary structure of ribonuclease F1, the guanine specific ribonuclease from Fusarium moniliforme, has been determined. Ribonuclease F1 consists of 106 amino acid residues and has a molecular weight of 10,980. It has a pyroglutamyl residue at the N-terminus. Comparison of the primary structures of four fungal ribonucleases so far sequenced shows that the amino acid sequences around the assumed active site residues are indeed well conserved. However, structurally important half cystine residues are arranged variously.
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PMID:The primary structure of ribonuclease F1 from Fusarium moniliforme. 643 32

The three-dimensional structure of Ribonuclease St (RNase St), the extracellular ribonuclease from Streptomyces erythreus, has been deduced based on a preliminary electron density map at 2.5 A resolution. RNase St has a substrate specificity similar to ribonuclease T1 which catalyzes the splitting of the phosphodiester bond of guanylic acid. Crystals grown as diamond plates have space group C2 with unit cell parameters a=88.4, b=33.0, c=69.0 A, beta = 98.4 degrees having two enzyme molecules per asymmetric unit. Phases were obtained by use of KAu(CN)4, phenylmercuric acetate and UO2 (CH3COO)2. The overall dimensions of the molecule are 40 X 30 X 25 A. The most prominent secondary structural features are two turns of alpha-helix and a three strand stretch of antiparallel beta-sheet. The alpha-carbon backbone of RNase St seems to have no apparent correlation with that of ribonuclease A.
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PMID:Preliminary crystal structure analysis of a microbial, guanine-specific ribonuclease St at 2.5 A resolution. 679 45

To address a number of conflicting reports in the literature, we undertook an infrared spectroscopic study to test for the presence of native-like secondary structures in thermally denatured ribonuclease A. Ribonuclease A does not aggregate at high temperatures, and the infrared spectrum shows a completely featureless amide I band contour. Using 13C-labeled urea, we were also able to obtain the infrared spectrum of the chemically denatured protein, which is practically identical with that of the heat-denatured protein. To the best of our knowledge, this is the first study that uses 13C-labeled urea as a chemical denaturant which circumvents the problem encountered with the strong absorption of urea in the conformation-sensitive amide I region of proteins; it opens up the possibility of investigating protein folding/unfolding processes in the presence of high concentrations of chemical denaturants. From an analysis of the amide I region of the infrared spectra of thermally and chemically denatured RNase A, it was concluded that heat-denatured ribonuclease A does not contain any significant amount of authentic hydrogen-bonded secondary structures. Furthermore, a comparison of the infrared spectra of ribonuclease A with those of ribonuclease T1 demonstrates that in spite of major differences between their native structures there are practically no differences between their heat-denatured states. This would not be expected if there were residual native-like secondary structures in the thermally denatured state of one or both of these proteins.
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PMID:Ribonuclease A revisited: infrared spectroscopic evidence for lack of native-like secondary structures in the thermally denatured state. 757 55

Ribonuclease T1 (RNase T1) carboxymethylated at the gamma-carboxyl group of Glu-58 with iodoacetic acid is known to be completely inactive while it retains an almost full substrate-binding ability. In order to further clarify the effects of the carboxymethylation, the thermal stabilities of intact and Glu-58-carboxymethylated (CM-) RNase T1 were compared by measuring 1H NMR spectra at various temperatures. The transition curves of unfolding were obtained by plotting, as a function of temperature, the peak areas for the alpha and delta protons of Asn-81 and Ile-90, respectively, which are well apart from each other in the three-dimensional structure of the enzyme. For each of intact and CM-RNase T1, the transition curve of the Asn-81 alpha proton was identical with that of the Ile-90 delta methyl protons, suggesting that the thermal unfolding occurred simultaneously in every part of the molecule of CM-RNase T1 as well as of intact RNase T1. The midpoint of unfolding was 52 degrees C for intact RNase T1, and was increased by 9 degrees C upon carboxymethylation at Glu-58. This marked stabilization by carboxymethylation is thought to be due to formation of a salt bridge between the introduced carboxymethyl group and the neighboring guanidium group of Arg-77.
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PMID:Thermal stabilization of ribonuclease T1 by carboxymethylation at Glu-58 as revealed by 1H nuclear magnetic resonance spectroscopy. 791 96

Our recent equilibrium dialysis studies showed that proteins are able to interact preferentially with acrylamide (Punyiczki et al. (1993) Biophys. Chem. 47, 9-19). The presence of considerable amounts of acrylamide--albeit weakly bound--in the protein volume, coupled with the failure of a simple gating model of quenching to rationalise viscosity dependence of the quenching of tryptophan (Trp) fluorescence in Ribonuclease T1 (RNase T1) has prompted us to explore a new model, the two-phase model for quenching. According to this model, the dynamic quenching is accomplished by quencher molecules already in the protein phase at the moment of excitation. Some of the molecules may, at this moment, form an encounter complex with the fluorophore and thus be responsible for the observed static contribution. We use the rate equation derived from our model to study the viscosity dependence of acrylamide quenching of Trp fluorescence in RNase T1. The model allows us to separate co-solvent effects: the chemical effect on the protein and on the distribution of quencher molecules between the bulk and the protein phases and, further, the viscosity effect due to coupling between the bulk viscosity and the local friction affecting intramolecular fluctuations of the protein matrix. We express local friction in terms of bulk viscosity, eta, and a coupling constant kappa (friction = eta kappa). Addition of glycerol up to 65% is characterised by a kappa of 0.50. The viscosity dependence of the apparent bimolecular quenching constant is a combination of two compensating effects: changes in chemical activity and changes in patterns of structural fluctuations.
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PMID:Coupling between external viscosity and the intramolecular dynamics of ribonuclease T1: a two-phase model for the quenching of protein fluorescence. 794 83

Guanidinium chloride is a commonly used denaturant to unfold native proteins and to determine their Gibbs free energy of stabilization, delta Gstab. Here we show that this denaturant has a dual role for the stability and the folding of the model protein ribonuclease T1. When present at low concentration (0-0.3 M), guanidinium chloride stabilizes the folded protein toward thermal and urea-induced unfolding and decreases the rate of unfolding. At high concentration the function of guanidinium chloride as a denaturant dominates and ribonuclease T1 is cooperatively unfolded. Ribonuclease T1 is also strongly stabilized by other salts, such as NaCl, at low concentrations, and the dependence of the thermal stability on salt concentration is not linear. Such a complex behavior was not found in control experiments with pancreatic ribonuclease A. The stabilization in the presence of low concentrations of guanidinium chloride originates probably from the binding of guanidinium ions to one or a few cation binding sites that exist in native ribonuclease T1. It is not observed when an additional salt, NaCl, is present simultaneously. The favorable interaction of guanidinium chloride with the native protein leads to increased values for delta Gstab, when unfolding transitions induced by guanidinium chloride are analyzed on the basis of the two-state model by the linear extrapolation procedure. The noncoincidence of these delta Gstab values with stability data derived from urea-induced or thermal unfolding transitions does not imply that the two-state model is not appropriate but that the linear extrapolation to zero molar denaturant is incorrect.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stabilization of a protein by guanidinium chloride. 834 3

Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular permutations. The original amino-terminus and carboxyl-terminus of the mutant (C2, 10A) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent-exposed loops at Ser-35 (cp35S1), Asp-49 (cp49D1), Gly-70 (cp70G1), and Ser-96 (cp96S1). These circularly permuted RNase T1 mutants retained 35-100% of the original catalytic activity for the hydrolysis of guanylyl(3'-->5')cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild-type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild-type RNase T1 and 6.4 kcal/mol for (C2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight beta-turn between Glu-82 and Gln-85. New termini were inserted at Asn-83 (cp83N1), Asn-84 (cp84N1), and Gln-85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.
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PMID:Are turns required for the folding of ribonuclease T1? 874 97

Ribonuclease T1 (RNase T1) specifically cleaves RNA at guanylyl residues. Previous studies revealed the presence of an enzyme-subsite interaction for adenosine residues of ApGpC and ApGpU substrates (Osterman and Walz (1979) Biochemistry 10, 1984-1988). The binding of ApG and 2'-deoxyadenylyl-(3',5')-guanosine (dApG) with RNase T1 was studied in the pH range 5-9 using ultraviolet difference spectroscopy. The association constants for these dinucleoside monophosphates showed the same pH dependence both of which differed at low pH values with that for the methyl phosphoester of 5'-GMP (MepG). This difference suggested that binding of the adenosine group is strongly dependent on the deprotonation of an enzyme/ligand group with a pKa value of < or = 4.8. delta G zero for ApG binding minus that for MepG at pH > 6 yielded a delta delta G of -1.17 +/- 0.10 kcal/mol which is a measure of the contribution of the adenosine moiety to binding. ApG bound more tightly than dApG with a mean delta delta G value of -0.73 +/- 0.10 kcal/mol which demonstrated the involvement of the adenosine 2'-OH group in binding. These and other comparisons indicated that delta delta G for maximal binding the adenine base per se was -0.44 kcal/mol. delta delta G for binding pdApdG minus that for dApdG (-0.94 kcal/mol) suggested an enzyme subsite for the phosphomonoester group of former ligand.
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PMID:Upstream subsite interactions for oligonucleotide binding with ribonuclease T1. 904 88


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