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)

Using point mutated overproducing strains of E. coli, ribonuclease T1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp, Tyr45Trp or Trp59Tyr and the corresponding double substitutions Tyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr. Steady state kinetics of the transesterification reaction for the two dinucleoside monophosphate substrates guanylyl-3',5'-cytidine and guanylyl-3',5'-adenosine indicate that the tryptophan can be introduced in different positions within the ribonuclease T1 molecule without abolishing enzymatic activity. The Trp59Tyr exchange even enhances catalysis of the cleavage reaction (kcat/Km) relative to the wild type enzyme and similar effects are found with single tyrosine to tryptophan substitutions. For the pH dependencies of the guanylyl-3',5'-cytidine transesterification reaction of wild type ribonuclease T1 and of the variants, typically bell-shaped curves are observed with a plateau in the range pH 4.5-7.0. Their shapes and slopes indicate that the enzymes are comparable in their macroscopic pKa values. At pH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-fold higher transesterification activity for guanylyl-3',5'-adenosine and a 2-fold increase for guanylyl-3',5'-cytidine compared to the wild type enzyme, i.e. this variant catalyses the transesterification of the substrate guanylyl-3',5'-adenosine with the same or better efficiency as guanylyl-3',5'-cytidine.
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PMID:Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45. 824 97

Tryptophan fluorescence intensity decay in proteins is modeled by multiexponential functions characterized by lifetimes and preexponential factors. Commonly, multiple conformations of the protein are invoked to explain the recovery of two or more lifetimes from the experimental data. However, in many proteins the structure seems to preclude the possibility of multiple conformers sufficiently different from one another to justify such an inference. We present here another plausible multiexponential model based on the assumption that an energetically excited donor surrounded by N acceptor molecules decays by specific radiative and radiationless relaxation processes, and by transferring its energy to acceptors present in or close to the protein matrix. If interactions between the acceptors themselves and back energy transfer are neglected, we show that the intensity decay function contain 2N exponential components characterized by the unperturbed donor lifetime, by energy transfer rates and a probability of occurrence for the corresponding process. We applied this model to the fluorescence decay of holo- and apoazurin, ribonuclease T1, and the reduced single tryptophan mutant (W28F) of thioredoxin. Use of a multiexponential model for the analysis of the fluorescence intensity decay can therefore be justified, without invoking multiple protein conformations.
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PMID:A model for multiexponential tryptophan fluorescence intensity decay in proteins. 831 71

In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C. Temperature effects were interpreted by proposing that solvent damping of internal protein motions is particularly effective for low frequency, large amplitude, structural fluctuations yielding highly flexible conformers of the macromolecule.
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PMID:Glycerol effects on protein flexibility: a tryptophan phosphorescence study. 836 22

The reverse micellar system formed by the negatively charged surfactant AOT and the organic solvent isooctane is used to solubilize the protein RNase T1. The physicochemical properties of the entrapped protein have been studied using intrinsic tryptophan fluorescence and far-and near-UV CD. These studies indicate a similar structure for the protein in reverse micelles and in pH 7.0 buffer. Thermal unfolding has been studied as a function of W0, the molar ratio of water to AOT, in the solution. Measuring the change in fluorescence intensity as a function of temperature, we observe a reversible transition for W0 in the range 5-12. Heating rate dependencies carried out on these transitions (0.6-3.0 degrees C/min) indicate that the transition temperature and the apparent van't Hoff enthalpy change depend on the scanning rate as well as on W0. The values of the transition temperature, T(m) and the enthalpy change, delta H degrees(un), extrapolated to an infinitely slow scanning rate, are analyzed considering the electrostatic interaction of the charged residues of the protein with the charges of the surfactant molecules forming reverse micelles, the variation of the size of the reverse micelles, and the relative rates of unfolding, refolding, and irreversible denaturation.
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PMID:Reversible thermal unfolding of ribonuclease T1 in reverse micelles. 867 44

Time-resolved fluorescence study of single tryptophan-containing proteins, nuclease, ribonuclease T1, protein G, glucagon, and mastoparan, has been carried out. Three different methods were used for the analysis of fluorescence decays: the iterative reconvolution method, as reviewed and developed in our laboratory, the maximum entropy method, and the recent method that we called "energy transfer" method. All the proteins show heterogeneous fluorescence kinetics (multiexponential decay). The origin of this heterogeneity is interpreted in terms of current theories of electron transfer process, which treat the electron transfer process as a radiationless transition. The theoretical electron transfer rate was calculated assuming the peptide bond carbonyl as the acceptor site. The good agreement between experimental and theoretical electron-transfer rates leads us to suggest that the electron-transfer process is the principal quenching mechanism of Trp fluorescence in proteins, resulting in heterogeneous fluorescence kinetics. Furthermore, the origin of apparent homogeneous fluorescence kinetics (monoexponential decay) in some proteins also can be explained on the basis of electron-transfer mechanism.
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PMID:On the involvement of electron transfer reactions in the fluorescence decay kinetics heterogeneity of proteins. 1156 1

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.
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PMID:Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa. 1537 18

An important feature of tryptophan phosphorescence, crucial for probing protein structure and dynamics, is the drastic reduction of the lifetime (tau) in fluid solutions. Initial reports of indole and derivatives showed that tau decreases from 6 s in rigid glasses to about 1 ms in aqueous solutions at ambient temperature. Recently a report by Fischer et al. questioned the validity of the millisecond lifetime, claiming that in millimolar electrolyte solutions tau is about 40 micros, similar to the 12-30 micros of earlier determinations based on flash photolysis. Longer lived phosphorescence was detected in pure water but because it exhibited an initial growing phase and an anomalously large triplet yield, the emission was attributed to an artifact arising from the slow, first-order, geminate recombination of the radical cation and electron generated by photochemistry. In this study, we reexamine both the phosphorescence lifetime and the triplet quantum yield of indole, N-acetyl tryptophanamide (NATA), N-methyl tryptophan and the tryptophan-glycine-glycine tripeptide under the same conditions adopted by Fischer et al. as well as over a wider range of electrolyte and buffering salts concentrations, pH, solvent and temperature. Throughout, the results show that the phosphorescence decay is slow and uniform down to the 12 micros resolution of the instrument, with no evidence of short-lived, 40 micros-like components. Most compelling was the similarity between the fluorescence-normalized triplet yield of indole derivatives in water and that of W59 in the protein ribonuclease T1 or of NATA in rigid glasses. Its invariance over experimental conditions that varied the production of photoproducts several fold and the characteristic susceptibility of the triplet lifetime to O2, proton and ground state quenching demonstrated that the triplet state was formed predominantly through normal intersystem crossing and that its unquenched lifetime was at least 9 ms.
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PMID:The triplet-state lifetime of indole derivatives in aqueous solution. 1562 31

Ribonuclease T1 is an enzyme that cleaves single-stranded RNA with high specificity after guanylyl residues. Although this enzyme is a very good characterized protein with respect to structure and enzymatic function, we were only recently successful in generating RNase T1-RV, a variant where the specificity was changed from guanine to purine. As this change of substrate specificity was made at the cost of activity, the aim was now to further improve the overall activity of the enzyme. Therefore, we have substituted the tryptophan in position 59 by tyrosine. This substitution led to an increase of enzymatic activity in comparison to variant RV to 425%. As the extent of this enhancement is unique so far we have crystallized and analyzed the structure of this variant in order to get more insights into the reasons for this. Here, we present the crystal structure of this so-called RNase T1-R2 at 2.1A resolution. The structure was determined by molecular replacement using the coordinates of the RV variant (PDB entry: 1Q9E). The data were refined to an R-factor of 18.7% and R(free) of 24%, respectively. The asymmetric unit contains three molecules and the crystal packing is very similar to that of variant RV.
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PMID:Purine activity of RNase T1RV is further improved by substitution of Trp59 by tyrosine. 1615 2

This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA with four carbonyls, to 28,500 for AAWAA with six carbonyls. This suggests that the four carbonyls of AWA are better able to quench Trp fluorescence than the six carbonyls of AAWAA, and this must reflect a difference in the conformations of the peptides. For the pentapeptides, EAWQE has a fluorescence intensity that is more than 50% greater than DYWTG, showing that the amino acid sequence influences the fluorescence intensity either directly through side-chain quenching and/or indirectly through an influence on the conformational ensemble of the peptides. Our results show that peptides are generally better models for the Trp residues in proteins than NATA. Finally, our results emphasize that we have much to learn about Trp fluorescence even in simple compounds.
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PMID:Peptide sequence and conformation strongly influence tryptophan fluorescence. 1806 77

The origin of the biexponential fluorescence decay of Trp in ribonuclease T1 under mildly destabilizing conditions, such as increased pH or temperature, or the presence of detergent, is still not understood. We have performed two extended replica-exchange molecular dynamics simulations to obtain a detailed representation of the native state at two protonation states corresponding to a high and low pH. At high pH, the appearance of partially unfolded states is evident. We found that this pH-induced destabilization originates from increased global repulsion as well as reduced local favorable electrostatic interactions and reduced H-bonding strength of His(27), His(40), and His(92). At high pH, alternative tryptophan rotamers appear and are linked to a distorted environment of the tryptophan, which also acts as a separate source of ground-state heterogeneity. The total population of these alternative conformations agrees well with the amplitude of the experimentally observed secondary fluorescence lifetime.
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PMID:Tryptophan conformations associated with partial unfolding in ribonuclease T1. 1975 84


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