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)

The carboxymethylation of RNase T1 at the gamma-carboxyl group of Glu58 leads to a complete loss of the enzymatic activity while it retains substrate-binding ability. Accompanying the carboxymethylation, RNase T1 undergoes a remarkable thermal stabilization of 9 degrees C in the melting temperature (Tm). In order to clarify the inactivation and stabilization mechanisms of RNase T1 by carboxymethylation, the crystal structure of carboxymethylated RNase T1 (CM-RNase T1) complexed with 2'-GMP was determined at 1.8 A resolution. The structure, including 79 water molecules and two Na+, was refined to an R factor of 0.194 with 10 354 reflections > 1 sigma (F). The carboxyl group of CM-Glu58, which locates in the active site, occupies almost the same position as the phosphate group of 2'-GMP in the crystal structure of intact RNase T1.2'-GMP complex. Therefore, the phosphate group of 2'-GMP cannot locate in the active site but protrudes toward the solvent. This forces 2'-GMP to adopt an anti form, which contrasts with the syn form in the crystal of the intact RNase T1.2'-GMP complex. The inaccessibility of the phosphate group to the active site can account for the lack of the enzymatic activity in CM-RNase T1. One of the carboxyl oxygen atoms of CM-Glu58 forms two hydrogen bonds with the side-chains of Tyr38 and His40. These hydrogen bonds are considered to mainly contribute to the higher thermal stability of CM-RNase T1. Another carboxyl oxygen atoms of CM-Glu58 is situated nearby His40 and Arg77. This may provide additional electrostatic stabilization.
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PMID:Crystal structure of ribonuclease T1 carboxymethylated at Glu58 in complex with 2'-GMP. 867 90

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.
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PMID:Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange. 923 39

During the last decade, protein engineering has been used to identify the residues that contribute to the ribonuclease-T1-catalyzed transesterification. His40, Glu58 and His92 accelerate the associative nucleophilic displacement at the phosphate atom by the entering 2'-oxygen downstream guanosines in a highly cooperative manner. Glu58, assisted by the protonated His40 imidazole, abstracts a proton from the 2'-oxygen, while His92 protonates the leaving group. Tyr38, Arg77 and Phe100 further stabilize the transition state of the reaction. A functionally independent subsite, including Asn36 and Asn98, contributes to chemical turnover by aligning the substrate relative to the catalytic side chains upon binding of the leaving group. An invariant structural motive, involving residues 42-46, renders ribonuclease T1 guanine specific through a series of intermolar hydrogen bonds. Tyr42 contributes significantly to guanine binding through a parallel face-to-face stacking interaction. Tyr45, often referred to as the lid of the guanine-binding site, does not contribute to the binding of the base.
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PMID:A decade of protein engineering on ribonuclease T1--atomic dissection of the enzyme-substrate interactions. 924 2

His92 of Ribonuclease T1 combines functional and structural features involving both imidazole nitrogens. To evaluate the use of Asn and Gln substitutions in dissecting the properties of histidines, we analysed the consequences of the His92Gln and His92Asn substitutions on the enzyme's structure, function, and conformational stability by protein engineering and X-ray crystallographic methods. In the X-ray structures of wild-type and His92Gln RNase T1 in complex with 2'-GMP the His92-N epsilon 2 and Gln92-N epsilon 2 atoms are isosterically equivalent. Similarly, the His92N delta 1H...OAsn99 hydrogen bond observed in wild-type is replaced by an equivalent Asn92N delta 2H...OAsn99 in the His92Asn mutant structure. Double mutant cycles at a single position were used to analyse the intermolecular and intramolecular interactions of the exchangeable proton and the individual histidine nitrogens. Urea denaturation measurements as a function of pH revealed that the exchangeable proton of His92, rather than its imidazole ring is contributing about 1 kcal/mol to the conformational stability of RNase T1. The stabilizing and the destabilizing effects of the (His-->Gln) and the (His-->Asn) mutations on urea denaturation of RNase T1 at pH 9.0 suggest that the unprotonated N delta 1 and N epsilon 2 atoms contribute in a compensating way to the conformational stability of RNase T1. A comparative study of the kinetics of all mutants suggests that the protonated His92 imidazole is a strictly co-operative catalytic device.
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PMID:Dissecting histidine interactions of ribonuclease T1 with asparagine and glutamine replacements: analysis of double mutant cycles at one position. 946 38

We used mutants of RNase T1 and the Rp isomer of a thiosubstituted substrate to determine stereospecific thioeffects on catalysis. The analysis reveals subtle structural and functional changes in the intermolecular transition state interactions. Tyr 38 contributes to catalysis through a hydrogen bond with the pro-Rp oxygen. Y38F RNase T1 prefers the Rp thiosubstituted analog over the natural phosphodiester substrate that is favored by the wild type enzyme.
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PMID:An engineered ribonuclease preferring phosphorothioate RNA. 958 98

The hydration of uncomplexed RNase T1 was investigated by NMR spectroscopy at pH 5.5 and 313 K. Two-dimensional heteronuclear NOE and ROE difference experiments were employed to determine the spatial proximity and the residence times of water molecules at distinct sites of the protein. Backbone carbonyl oxygens involved in intermolecular hydrogen bonds to water molecules were identified based on 1J(NC) coupling constants. These coupling constants were determined from 2D-H(CA)CO and 15N-HSQC experiments with selective decoupling of the 13C alpha nuclei during the t1 evolution time. Our results support the existence of a chain of water molecules with increased residence times in the interior of the protein which is observed in several crystal structures with different inhibitor molecules and serves as a space filler between the alpha-helix and the central beta-sheet. The analysis of 1J(NC) coupling constants demonstrates that some of the water molecules seen in crystal structures are not involved in hydrogen bonds to backbone carbonyls as suggested by crystal structures. This is especially true for a water molecule, which is probably hydrogen bonded by the protonated carboxylate group of D76 and the hydroxyl group of T93 in solution, and for a water molecule, which was reported to connect four different amino acid residues in the core of the protein by intermolecular hydrogen bonds.
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PMID:Hydration water molecules of nucleotide-free RNase T1 studied by NMR spectroscopy in solution. 961 95

Ribonucleases Sa, Sa2, and Sa3 are three small, extracellular enzymes produced by different strains of Streptomyces aureofaciens with amino acid sequences that are 50% identical. We have studied the unfolding of these enzymes by heat and urea to determine the conformational stability and its dependence on temperature, pH, NaCl, and the disulfide bond. All three of the Sa ribonucleases unfold reversibly by a two-state mechanism with melting temperatures, Tm, at pH 7 of 48.4 degrees C (Sa), 41.1 degrees C (Sa2), and 47.2 degrees C (Sa3). The Tm values are increased in the presence of 0.5 M NaCl by 4.0 deg. C (Sa), 0.1 deg. C (Sa2), and 7.2 deg. C (Sa3). The Tm values are decreased by 20.0 deg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single disulfide bond in the molecules is reduced. We compare these results with similar studies on two other members of the microbial ribonuclease family, RNase T1 and RNase Ba (barnase), and with a member of the mammalian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the conformational stabilities of the ribonucleases are (kcal/mol): 2.9 (Sa2), 5.6 (Sa3), 6.1 (Sa), 6.6 (T1), 8.7 (Ba), and 9.2 (A). Our analysis of the stabilizing forces suggests that the hydrophobic effect contributes from 90 to 110 kcal/mol and that hydrogen bonding contributes from 70 to 105 kcal/mol to the stability of these ribonucleases. Thus, we think that the hydrophobic effect and hydrogen bonding make large but comparable contributions to the conformational stability of these proteins.
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PMID:Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3. 963 16

We present the spatial structure of binase, a small extracellular ribonuclease, derived from 1H-NMR* data in aqueous solution. The total of 20 structures were obtained via torsion angle dynamics using DYANA program with experimental NOE and hydrogen bond distance constraints and phi and chi1 dihedral angle constraints. The final structures were energy minimised with ECEPP/2 potential in FANTOM program. Binase consists of three alpha-helices in N-terminal part (residues 6-16, 26-32 and 41-44), five-stranded antiparallel beta-sheet in C-terminal part (residues 51-55, 70-75, 86-90, 94-99 and 104-108) and two-stranded parallel beta-sheet (residues 22-24 and 49-51). Three loops (residues 36-39, 56-67 and 76-83), which play significant role in biological functioning of binase, are flexible in solution. The differences between binase and barnase spatial structures in solution explain the differences in thermostability of binase, barnase and their hybrids.
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PMID:Three-dimensional structure of binase in solution. 970 13

We report the 1.7 A resolution structure of RNase Sa complexed with the polypeptide inhibitor barstar. The crystals are in the hexagonal space group P65 with unit-cell dimensions a = b = 56.9, c = 135.8 A and the asymmetric unit contains one molecule of the complex. RNase Sa is an extracellular microbial ribonuclease produced by Streptomyces aureofaciens. Barstar is the natural inhibitor of barnase, the ribonuclease of Bacillus amyloliquefaciens. It inhibits RNase Sa and barnase in a similar manner by steric blocking of the active site. The structure of RNase Sa is very similar to that observed in crystals of the native enzyme and its complexes with nucleotides. Barstar retains the structure found in its complex with barnase. The accessible surface area of protein buried in the complex is about 300 A2 smaller and there are fewer hydrogen bonds in the enzyme-inhibitor interface in RNase Sa-barstar than in barnase-barstar, providing an explanation of the reduced binding affinity in the former. Previous studies of barstar complexes have used mutants of the inhibitor and this is the first structure which includes wild-type barstar.
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PMID:Recognition of RNase Sa by the inhibitor barstar: structure of the complex at 1.7 A resolution. 975 10

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.
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PMID:Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1. 981 11


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