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 tryptophan fluorescence of two membrane proteins (outer membrane protein A and lactose permease), a 21-residue hydrophobic peptide, three soluble proteins (rat serum albumin, ribonuclease T1, and azurin), and N-acetyltryptophanamide (NATA) was investigated by time-resolved measurements extended over 65 ns. A long lifetime component with a characteristic time of 25 ns and an amplitude below 1% was found for outer membrane protein A, lactose permease, the peptide in lipid membranes, and azurin in water, but not for rat serum albumin, ribonuclease T1, and NATA in water. When outer membrane protein A was dissolved and unfolded in guanidinum hydrochloride, the long lifetime component disappeared. Hence, a hydrophobic environment seems to be a necessary requirement for the long lifetime component to be present. However, NATA dissolved in butanol does not exhibit the long lifetime component, while the peptide dissolved in the same solvent under conditions which preserve its helical structure does show the long lifetime. Thus, a regular secondary structure for the polypeptide chain to which the tryptophan residue belongs seems to be a second necessary requirement for the long lifetime component to be present. The long lifetime component may therefore be seen in the context of protein substates.
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PMID:A long lifetime component in the tryptophan fluorescence of some proteins. 772 67

Disulfide bonds in a folding protein chain are equivalent to prematurely formed native-like tertiary interactions. We investigated whether the mechanism of protein folding is changed by the presence of disulfide bonds. As a model we used the S54G/P55N-variant of ribonuclease T1, a protein with two disulfide bonds and a single cis proline (Pro39), and we measured both the direct and the proline-limited folding reactions before and after breaking of the disulfide bonds. The folding kinetics were compared under refolding conditions, in the regions of the urea-induced unfolding transitions of the two forms, and under unfolding conditions. The kinetics in the transition regions were analyzed on the basis of a three-species mechanism and all microscopic rate constants of folding and of prolyl isomerization could be determined as a function of the urea concentration from the measured rates and amplitudes. These kinetic analyses indicated that the disulfide bonds can be rather unfavorable for the folding of S54G/P55N-ribonuclease T1. Under strongly native conditions they retard the rate-limiting trans-->cis isomerization of Pro39 because they allow the rapid formation of partially ordered structure prior to the proline-limited refolding reaction. Under unfolding conditions the isomerization of Pro39 is not affected. The direct unfolding and refolding reactions in the transition region of polypeptide chains with correct prolyl isomers are also decelerated when the disulfide bonds are present. These changes in the folding kinetics are possibly related to the decrease in chain flexibility that is caused by the disulfide bonds. A high flexibility is probably important throughout folding, and in the case of ribonuclease T1 a premature locking of tertiary contacts by intact disulfide bonds can interfere unfavorably with both the direct and the proline-limited folding reactions.
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PMID:Intact disulfide bonds decelerate the folding of ribonuclease T1. 801 91

We undertook a detailed comparative analysis of the infrared spectra of wild-type ribonuclease T1 and three mutants: two single mutants, Tyr-45-->Trp (Y45W) and Trp-59-->Tyr (W59Y), and a double mutant, Tyr-45-->Trp/Trp-59-->Tyr (Y45W/W59Y). These mutants were selected because they are known to affect the activity of the enzyme. The structural differences were evaluated by using peptide backbone and side-chain "marker" bands as conformation-sensitive monitors. All mutations lead to a decrease of the thermal transition temperature, though the mutation Tyr-45-->Trp affects the Tm to a lesser degree than the replacement of Trp-59 by Tyr, both in the single (W59Y) and in the double (Y45W/W59Y) mutant. Small changes in the protein backbone conformation and in the microenvironment of certain amino acids, induced by the point mutations, could be detected. In particular, we found subtle differences in the hydrogen bonding pattern of the beta-strands in the mutants W59Y and Y45W/W59Y, compared to that in wild-type RNase T1 and in the mutant Y45W. Practically identical spectra in the amide I region were obtained for the double mutant Y45W/W59Y and the single mutant W59Y, demonstrating that it is the change from Trp to Tyr in position 59 (located at the interface between the alpha-helix and a beta-strand) which affects the overall protein conformation. The mutation Tyr to Trp in position 45, on the other hand, has practically no impact on the polypeptide backbone conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Impact of point mutations on the structure and thermal stability of ribonuclease T1 in aqueous solution probed by Fourier transform infrared spectroscopy. 807 73

Alterations in flexibility of monomeric proteins induced by hydrostatic pressure in the predenaturational range (< or = 3 kbar) were probed through the decay kinetics of tryptophan phosphorescence. With apoazurin, ribonuclease T1, wild-type and V67G mutant and phosphoglycerate kinase, pressure effects on the triplet lifetime (tau) and the amplitudes of multicomponent decays emphasize that subtle changes in conformation are ubiquitous. With apoazurin the increase in tau attests to a tightening of the protein core that is enhanced at high temperature. On the contrary, tau decreases with ribonuclease T1, wild-type and mutant, and with phosphoglycerate kinase, indicating that pressure induces a greater flexibility to protein regions in proximity to the surface of the macromolecule. For phosphoglycerate kinase the decrease in tau and the parallel increase in fluorescence intensity and red-shift of the fluorescence spectrum unveil an "unfolding" like transition with midpoint pressures of 1.1 kbar at 5 degrees C and 1.6 kbar at 25 degrees C. Evidence that unfolding of the C-domain of this protein is, however, less than complete is provided by a delta G zero that is about half of that obtained by denaturation in guanidine hydrochloride and also by the ability of this structure to undergo conformational drift. In 70% glycerol, pressure effects on tau of apoazurin are attenuated while for ribonuclease T1 there is a reversal of the tendency with a pronounced increase in tau. With phosphoglycerate kinase glycerol abolishes entirely the "unfolding" transition and all hysteresis effects. A consistent picture of these findings is provided in terms of the location of the probe and of the opposing effects that pressure exerts on protein flexibility by reducing internal cavities and increasing the hydration of the polypeptide.
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PMID:Pressure effects on protein flexibility monomeric proteins. 808 48

The ternary complex formed between RNase T1, guanosine 3',5'-bisphosphate (3',5'-pGp) and Pi crystallizes in the cubic space group I23 with a = 8.706(1) nm. In a previous publication [Lenz, A., Heinemann, U., Maslowska, M. & Saenger, W. (1991) Acta Crystallogr. B47, 521-527], the structure of the complex (in which Pi was not located) was described at a resolution of 0.32 nm. This is now extended to 0.19 nm with newly grown, larger crystals. Refinement with restrained least-squares converged at R = 17.8% for 8027 reflections with [Fo[ > or = 1 sigma ([Fo[); the final model comprises 120 water molecules. 3',5'-pGp is bound to RNase T1 in the anti form, with guanine in the specific recognition site; the 3'-phosphate protrudes into the solvent, and the 5'-phosphate hydrogen bonds with Lys41 O and Asn43 N4. A tetrahedral anion assigned as Pi occupies the catalytic site and hydrogen bonds to the side chains of Tyr38, Glu58, Arg77 and His92. The overall polypeptide fold of RNase T1 in the cubic space group does not differ significantly from that in the orthorhombic space group P2(1)2(1)2(1) except for changes < or = 0.2 nm in loop regions 69-72 and 95-98.
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PMID:Three-dimensional structure of the ternary complex between ribonuclease T1, guanosine 3',5'-bisphosphate and inorganic phosphate at 0.19 nm resolution. 842 41

The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold.
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PMID:Preferential binding of an unfolded protein to DsbA. 861 14

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

Oxygen quenching of protein phosphorescence and activation enthalpies for the structural fluctuations underlying O2 and acrylamide diffusion were determined for RNase T1, glyceraldehyde-3-phosphate dehydrogenase and beta-lactoglobulin, which have the phosphorescing residues located in relatively solvent-exposed and flexible regions of the polypeptide. The results, compared with those obtained for proteins characterised by a very rigid environment, established that kqO2 was directly correlated to the flexibility of the protein matrix surrounding the chromophore. While the migration of acrylamide was characterised by delta H(double dagger), which was strongly dependent on the fluidity of the structure about the Trp residue, the values of the activation enthalpies for the oxygen migration of all the proteins studied were rather similar, approximately 10 kcal mol(-1), in spite of the depth of the chromophore and the rigidity of its environment. The implications of these findings for the migration of small solutes inside proteins have been discussed.
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PMID:Oxygen and acrylamide quenching of protein phosphorescence: correlation with protein dynamics. 1103 66

The heat capacities (DeltaC(p,f)) for the temperature-induced folding of proteins: barnase, lysozyme T4, papain, trypsin, ribonuclease T1, chymotrypsin, lysozyme and ribonuclease A have been calculated from the change in solvent accessible surface area between the native state and extended polypeptide chain. To visualize the effect of disulfide cross-links on molar heat capacity, loops of varying number of alanine residues and extended alanine chains with terminal cystein are modeled. The difference in DeltaC(p) values between the extended state and the loop conformation of proteins is linearly related to the number of residues in the loop. Corrections to the heat capacity of folding (DeltaC(p,f)) are applied for proteins with cross-links based on this observation. There is good correlation between corrected values of DeltaC(p,f) and experimental values.
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PMID:Heat capacity of folding of proteins corrected for disulfide cross-links. 1205 96

The measurement of (15)N NMR spin relaxation, which reports the (15)N-(1)H vector reorientational dynamics, is a widely used experimental method to assess the motion of the protein backbone. Here, we investigate whether the (15)N-(1)H vector motions are representative of the overall backbone motions, by analyzing the temperature dependence of the (15)N-(1)H and (13)CO-(13)C(alpha) reorientational dynamics for the small proteins binase and ubiquitin. The latter dynamics were measured using NMR cross-correlated relaxation experiments. The data show that, on average, the (15)N-(1)H order parameters decrease only by 2.5% between 5 and 30 degrees C. In contrast, the (13)CO-(13)C(alpha) order parameters decrease by 10% over the same temperature trajectory. This strongly indicates that there are polypeptide-backbone motions activated at room temperature that are not sensed by the (15)N-(1)H vector. Our findings are at variance with the common crank-shaft model for protein backbone dynamics, which predicts the opposite behavior. This study suggests that investigation of the (15)N relaxation alone would lead to underestimation of the dynamics of the protein backbone and the entropy contained therein.
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PMID:Temperature dependence of anisotropic protein backbone dynamics. 1284 71


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