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

The crystal structure of the C82A mutant of barstar, the intracellular inhibitor of the Bacillus amyloliquefaciens ribonuclease barnase, has been solved to a resolution of 2.8 A. The molecule crystallizes in the space group I41 with a dimer in the asymmetric unit. An identical barstar dimer is also found in the crystal structure of the barnase-barstar complex. This structure of uncomplexed barstar is compared to the structure of barstar bound to barnase and also to the structure of barstar solved using NMR. The free structure is similar to the bound state, and there are no significant main-chain differences in the 27-44 region involved in barstar binding to barnase. The C82A structure shows significant differences from the average NMR structure, both overall and in the binding region. In contrast to the crystal structure, the NMR structure shows an unusually high packing value based on the occluded surface algorithm, indicating errors in the packing of the structure. We show that the NMR structures of homologous proteins generally show large differences in packing value, while the crystal structures of such proteins have very similar packing values, suggesting that protein packing density is not well determined by NMR.
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PMID:Discrepancies between the NMR and X-ray structures of uncomplexed barstar: analysis suggests that packing densities of protein structures determined by NMR are unreliable. 957 82

A new triple-resonance pulse sequence, 3D HNHCACO, is introduced and discussed, which identifies sequential correlations of the backbone nuclei (H alpha (i-1), C alpha (i-1), C(i-1), NH(i), N(i)) of doubly labeled proteins in H2O. The three-dimensional (3D) method utilizes a recording of 15N and 13C resonances in a single indirect time domain, the 13C' resonance in another indirect time domain, and detects both NH and H alpha protons. A bidirectional coherence transfer (NH(i) <--> N(i) <--> C(i-1) <--> C alpha (i-1) <--> H alpha (i-1)) is effectuated, resulting in a single high-resolution 3D spectrum that contains the frequencies of all five backbone nuclei. The experiment was applied to the 12.3 kDa ribonuclease from Bacillus intermedius (Binase).
J Biomol NMR 1998 Feb
PMID:High-resolution detection of five frequencies in a single 3D spectrum: HNHCACO--a bidirectional coherence transfer experiment. 967 93

The dynamic behavior of the ribonuclease inhibitor barstar has been investigated by molecular dynamics (MD) simulations in explicit water. Two 2.5 ns MD simulations were performed, and an ensemble of 25 000 structures was generated. This ensemble reproduces the solution structures and is consistent with the experimental structural restraints from NMR spectroscopy. Reorientation of the backbone NH bond vectors and side chain methyl groups was monitored by calculation of autocorrelation functions and the generalized S2 order parameters. Order parameters derived for motion in the approximately 100 ps time scale were compared with those obtained from NMR relaxation measurements. Consistent with experiment, the backbone NH bond vectors were relatively rigid. In contrast, the side chain methyl groups exhibited a wide dynamic range, from restricted motion comparable to that of the backbone to rapid unrestricted motion. The order parameters for the methyl groups correlate well with their spatial separation from the backbone and are residue-type dependent. Smaller S2axis values were observed for leucine methyl groups, in part due to side chain hopping between two predominant rotamers (g+t and tg-). Motions such as the flipping of aromatic rings and the hopping of leucine side chains were prevalent within the hydrophobic core, suggesting that the core is fluid-like with low energy barriers between native conformational substates. Thus, our studies suggest that the entropy of the native state can be significant and should not be discounted in thermodynamic considerations of protein folding. On the basis of our results, the side chain motion represents the primary source of the residual entropy of the native state and entropic considerations based solely on backbone dynamics would be incomplete.
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PMID:Barstar has a highly dynamic hydrophobic core: evidence from molecular dynamics simulations and nuclear magnetic resonance relaxation data. 969 64

The practical exploitation of the vast numbers of sequences in the genome sequence databases is crucially dependent on the ability to identify the function of each sequence. Unfortunately, current methods, including global sequence alignment and local sequence motif identification, are limited by the extent of sequence similarity between sequences of unknown and known function; these methods increasingly fail as the sequence identity diverges into and beyond the twilight zone of sequence identity. To address this problem, a novel method for identification of protein function based directly on the sequence-to-structure-to-function paradigm is described. Descriptors of protein active sites, termed "fuzzy functional forms" or FFFs, are created based on the geometry and conformation of the active site. By way of illustration, the active sites responsible for the disulfide oxidoreductase activity of the glutaredoxin/thioredoxin family and the RNA hydrolytic activity of the T1 ribonuclease family are presented. First, the FFFs are shown to correctly identify their corresponding active sites in a library of exact protein models produced by crystallography or NMR spectroscopy, most of which lack the specified activity. Next, these FFFs are used to screen for active sites in low-to-moderate resolution models produced by ab initio folding or threading prediction algorithms. Again, the FFFs can specifically identify the functional sites of these proteins from their predicted structures. The results demonstrate that low-to-moderate resolution models as produced by state-of-the-art tertiary structure prediction algorithms are sufficient to identify protein active sites. Prediction of a novel function for the gamma subunit of a yeast glycosyl transferase and prediction of the function of two hypothetical yeast proteins whose models were produced via threading are presented. This work suggests a means for the large-scale functional screening of genomic sequence databases based on the prediction of structure from sequence, then on the identification of functional active sites in the predicted structure.
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PMID:Method for prediction of protein function from sequence using the sequence-to-structure-to-function paradigm with application to glutaredoxins/thioredoxins and T1 ribonucleases. 971 46

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a lectin possessing potent cell cytotoxicity. It was isolated from the oocytes of Rana catesbeiana (bull frog). From analysis of an extensive set of 1H homonuclear 2D NMR spectra we have completed the resonance assignments. Determination of the three-dimensional structure was carried out with the program X-PLOR using a total of 951 restraints including 814 NMR-derived distances, 61 torsion angles, and 76 hydrogen bond restraints. In the resultant family of 15 best structures, selected from a total of 150 calculated structures, the root-mean-square deviation from the average structure for the backbone heavy-atoms involved in well-defined secondary structure is 0.48 A, while that for all backbone heavy-atoms is 0.91 A. The structure of RC-RNase consists of three alpha-helices and two triple-stranded anti-parallel beta-sheets and folds in a kidney-shape, very similar to the X-ray crystal structure of a homolo gous protein, onconase isolated from Rana pipiens. We have also investigated the interaction between RC-RNase and two inhibitors, cytidylyl(2'-->5')guanosine (2',5'-CpG) and 2'-deoxycytidylyl(3'-->5')-2'-deoxyguanosine (3',5'-dCpdG). Based on the ligand-induced chemical shift changes in RC-RNase and the NOE cross-peaks between RC-RNase and the inhibitors, the key residues involved in protein-inhibitor interaction have been identified. The inhibitors were found to bind in a "retro-binding" mode, with the guanine base bonded to the B1 subsite. The His103 residue was found to occupy the B state with the imidazole ring pointing away from the active site. The structure coordinates and the NMR restraints have been deposited in the Brookhaven Protein Data Bank (1bc4 and 1bc4mr, respectively).
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PMID:The solution structure of a cytotoxic ribonuclease from the oocytes of Rana catesbeiana (bullfrog). 976 86

A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b5, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This omega-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.
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PMID:A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis. 978 9

Two-dimensional NMR spectroscopy has been used to monitor the exchange of backbone amide protons in ribonuclease A (RNase A) and its subtilisin-cleaved form, ribonuclease S (RNase S). Exchange measurements at two different pH values (5.4 and 6.0) show that the exchange process occurs according to the conditions of the EX2 limit. Differential scanning calorimetry measurements have been carried out in 2H2O under conditions analogous to those used in the NMR experiments in order to determine the values of DeltaCp, DeltaHu and Tm, corresponding to the thermal denaturation of both proteins. For the amide protons of a large number of residues in RNase A, the free energies at 25 degreesC for exchange competent unfolding processes are much lower than the calorimetric denaturation free energies, thus showing that exchange occurs through local fluctuations in the native state. For 20 other protons, the cleavage reaction had approximately the same effect on the exchange rate constants than on the equilibrium constant for unfolding, indicating that those protons exchange by global unfolding. There is a good agreement between the residues to which these protons belong and those involved in the putative folding nucleation site identified by quench-flow NMR studies. The unfolding free energies of the slowest exchanging protons, DeltaGex, as evaluated from exchange data, are much larger than the calorimetric free energies of unfolding, DeltaGu. Given the agreement between DeltaDeltaGex(A-S), the difference in free energy from exchange for a given proton of the two proteins, and DeltaDeltaGu(A-S), the difference in the calorimetric free energy of the two proteins, the discrepancy indicates that the intrinsic exchange rates in the unfolded state of those protons cannot be approximated by those measured in short unstructured peptides and, consequently, exchange for those protons in RNase A and S must occur through a rather structured denatured state.
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PMID:Hydrogen exchange in ribonuclease A and ribonuclease S: evidence for residual structure in the unfolded state under native conditions. 987 34

Pressure denaturation of Escherichia coli ribonuclease HI (RNase HI) was studied by Fourier transform infrared (FTIR) and two-dimensional NMR spectroscopy at pD* 3.0 and 25 degrees C. A reversible transition in the pressure range of 0.1-1090 MPa was observed with second-derivative FTIR experiments. A cooperative and gradual denaturation, involving both the secondary and tertiary structures, was observed between 240 and 450 MPa. The two peaks at 1629 and 1652 cm(-1), due to beta-strands and alpha-helices, respectively, did not fully disappear after the denaturation, and are different from the spectra of the random coil peptides. The hydrogen-deuterium exchange rates of the individual backbone amide protons were determined by heteronuclear NMR combined with the pressure-jump technique at 500, 650, and 850 MPa. Although most of the amides protected in the native structure are also highly protected in the pressure-denatured state, the rate constants (0.048 +/- 0.007 min(-1)) for the amide protons at 500 MPa are similar regardless of their locations, which is an indication of the EX1 mechanism of hydrogen-deuterium exchange. The pressure-denatured state of RNase HI at 500 MPa represents a novel denatured state, which is different from a typical molten globule state at atmospheric pressure (0.1 MPa), from the viewpoint of the homogeneous rate constants. The observations at 650 MPa are essentially the same as those at 500 MPa. However, at 850 MPa, the amide exchange rates for the highly hydrophobic C-terminal half of alpha-helix I are significantly slower than those for the other part of the protein, which can be interpreted as a hydrophobic collapse centered at the C-terminal half of alpha-helix I.
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PMID:Pressure-denatured state of Escherichia coli ribonuclease HI as monitored by Fourier transform infrared and NMR spectroscopy. 992 68

Sso7d is a basic 7-kDa DNA-binding protein from Sulfolobus solfataricus, also endowed with ribonuclease activity. The protein consists of a double-stranded antiparallel beta-sheet, onto which an orthogonal triple-stranded antiparallel beta-sheet is packed, and of a small helical stretch at the C-terminus. Furthermore, the two beta-sheets enclose an aromatic cluster displaying a fishbone geometry. We previously cloned the Sso7d-encoding gene, expressed it in Escherichia coli, and produced several single-point mutants, either of residues located in the hydrophobic core or of Trp23, which is exposed to the solvent and plays a major role in DNA binding. The mutation F31A was dramatically destabilizing, with a loss in thermo- and piezostabilities by at least 27 K and 10 kbar, respectively. Here, we report the solution structure of the F31A mutant, which was determined by NMR spectroscopy using 744 distance constraints obtained from analysis of multidimensional spectra in conjunction with simulated annealing protocols. The most remarkable finding is the change in orientation of the Trp23 side chain, which in the wild type is completely exposed to the solvent, whereas in the mutant is largely buried in the aromatic cluster. This prevents the formation of a cavity in the hydrophobic core of the mutant, which would arise in the absence of structural rearrangements. We found additional changes produced by the mutation, notably a strong distortion in the beta-sheets with loss in several hydrogen bonds, increased flexibility of some stretches of the backbone, and some local strains. On one hand, these features may justify the dramatic destabilization provoked by the mutation; on the other hand, they highlight the crucial role of the hydrophobic core in protein stability. To the best of our knowledge, no similar rearrangement has been so far described as a result of a single-point mutation.
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PMID:A single-point mutation in the extreme heat- and pressure-resistant sso7d protein from sulfolobus solfataricus leads to a major rearrangement of the hydrophobic core. 1050 41

The systematic difference between T2 values obtained from CPMG and T1p experiments was observed for backbone 15N nuclei of bacterial ribonuclease barnase. Theoretical consideration suggests that the observed difference is caused by off-resonance effects of 180 degree pulses of the CPMG pulse train. Namely, at off-resonance conditions T1-dependent secondary echo coherence pathways considerably contribute to the signal decay in the CPMG experiment and result in systematic (up to 10%) offset-dependent overestimation of 15N T2 measured by the CPMG technique. Under certain circumstances off-resonance effects result in dependence of 15N T2 on CPMG frequency, which might be erroneously interpreted as conformational exchange on the millisecond time-scale. A procedure for numerical correction of 15N T2 (CPMG) data is proposed.
J Biomol NMR 2000 Jul
PMID:Off-resonance effects in 15N T2 CPMG measurements. 1095 30


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