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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the construction and testing of a structural model at the nucleotide level for conformation CH of the central hairpin of genomic RNA from coliphage Q beta. The model was developed with the computer program MFOLD using both optimal and suboptimal predictions. Structural information obtained by electron microscopic analysis of Kleinschmidt spreadings of Q beta RNA was used to guide the modeling. The model was tested in solution with three enzymatic probes: RNase T1, RNase T2, and RNase V1, as well as four chemical probes: dimethylsulfate, diethylpyrocarbonate, kethoxal and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT). The structural analyses in solution are consistent with the predicted structural model. The model is also supported by comparative structural analysis with the related coliphage SP. The model provides a structural basis for published biochemical and genetic studies implicating large, long-range structural features in the co-regulation of viral coat and replicase expression. In addition, we show that the read-through region of the viral protein A1 forms a separate structural domain, and we suggest that it functions as a nucleation site that participates in the folding and refolding of the molecule during replication and translation. In addition to the central hairpin, we have analyzed the structure of the viral coat initiation region. Our studies show that the entire region consists of small local hairpins and that 26 nucleotides immediately surrounding the coat initiation codon are single-stranded.
J Mol Biol 1993 Sep 20
PMID:A two-dimensional model at the nucleotide level for the central hairpin of coliphage Q beta RNA. 837 1

We have chosen two members of the microbial RNase family, barnase and binase, which have 85% identity (17 substitutions and 1 deletion) and almost identical three-dimensional structure, to study the evolution of protein stability. The 17 residues that differ are scattered throughout the molecule. Each of the 17 differing residues has been mutated independently and the effect on protein stability analysed. Each point mutation has an effect on protein stability that ranges from +1.1 to -1.1 kcal mol-1. These changes in energy are additive. There is no clear correlation between the type of mutation and the effect on protein stability. A multiple mutant having six of the single mutations that increase the stability of barnase is 3.3 kcal mol-1 more stable than wild type and has the same activity. There could be selective pressure to maintain proteins at a certain stability and, consequently, mutations that decrease stability tend to be counterbalanced by stabilizing mutations. Alternatively, there could simply be pressure to maintain stability above a certain level, and any further increases in stability need not be maintained during evolution. These results suggest a simple way to improve the stability of proteins: choose two homologous proteins that have high similarity, mutate individually all of the residues that differ between the two, and combine the mutations that increase the stability in a multiple mutant.
J Mol Biol 1993 Sep 20
PMID:Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. 837 5

RNase F1, a guanine-specific ribonuclease from Fusarium moniliforme, was crystallized in two different forms, in the absence of an inhibitor and in the presence of 2'GMP. The crystal structure of the RNase F1 free form was solved by the molecular replacement method, using the co-ordinates of the RNase T1 complex with 2'GMP, and was refined to a final R-factor of 18.7%, using the data extended to 1.3 A resolution. For the crystal structure of the RNase F1 complex with 2'GMP, the solution of the molecular replacement method was obtained on the basis of the co-ordinates of the RNase F1 free form, and was refined to a final R-factor of 16.8%, using the data up to 2 A resolution. The two crystal structures of the RNase F1 free form and the complex with 2'GMP are very similar to each other as reflected by a small root-mean-square displacement (r.m.s.d.) value of 0.43 A for all C alpha atoms. The main differences between the two structures are associated with binding of 2'GMP in the substrate recognition site in the loop between Tyr42 and Glu46. A structural comparison between RNase F1 and RNase T1 shows a substantial similarity between all the C alpha atoms, as evidenced by a r.m.s.d. value of 1.4 A. The loop from residues 32 to 38 was strikingly different between these two enzymes, in both its conformation and its hydrogen bonding schemes. The side-chain of a catalytically active residue, His92, is shifted away from the catalytic site in RNase F1 by 1.3 A and 0.85 A with respect to the corresponding positions in the RNase T1 free form and in the RNase T1 complex with 2'GMP, respectively. In the RNase F1 complex, the guanine base of 2'GMP has a syn conformation about the glycosyl bond, and the furanose ring assumes a 3'-exo pucker, which is different from that found in the complex with RNase T1. In the catalytic site of the RNase F1 complex with 2'GMP, one water molecule was observed, which bridges the phosphate oxygen atoms of 2'GMP and the side-chains of the catalytically important residues, His92 and Arg77, through hydrogen bonds. A water molecule occupying the same position was found in the RNase F1 free form. The significance of this water molecule in the hydrolytic reaction is discussed.
J Mol Biol 1993 Apr 05
PMID:Crystal structures of ribonuclease F1 of Fusarium moniliforme in its free form and in complex with 2'GMP. 838 73

We report on the functional interplay between the His40, Glu58 and His92 catalysts of ribonuclease T1. The kinetic properties of the single His40Ala, Glu58Ala and His92Gln mutants have been compared with those of the corresponding double and triple mutants. When His40, Glu58 and His92 are mutated separately or together, we observe large effects on turnover but only minor effects on substrate binding. The free energy barriers to kcat introduced by the single His40Ala, Glu58Ala and His92Gln mutations are non-additive in the corresponding His40Ala + Glu58Ala, Glu58Ala + His92Gln and His40Ala + His92Gln double mutants; a significant dependence of the pairwise interactions on the third residue has been observed. Using a pair of related triple mutant boxes, we were able to divide the apparent coupling energies as calculated from the kinetic parameters of the various mutants either quantitatively or qualitatively into terms that measure intrinsic interaction energies between His40, Glu58 and His92 in wild-type enzyme and terms that account for a change in reaction mechanism that is associated with the Glu58Ala single mutation. In most cases, non-additivity may be explained by changes in enzyme mechanism. Apart from this change in mechanism, functional interactions have to be considered between His40 and Glu58, Glu58 and His92 as well as between His40 and His92 to explain non-additivity in all double-mutant cycles. It is concluded that the collaborative effects of His40, Glu58 and His92 decrease the energetic barrier to kcat by 6.8 kcal/mol. The overall effect caused by the triple mutation is smaller than that expected from the product of the fractional kcat values resulting from the individual mutations (11.0 kcal/mol), illustrating the limitations of using single mutants to probe the energetics of a catalytic group whose function is dependent upon interactions with others.
J Mol Biol 1993 Feb 05
PMID:Functional interactions among the His40, Glu58 and His92 catalysts of ribonuclease T1 as studied by double and triple mutants. 843 70

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase, in the pH region 2-6 have been determined. Barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as the pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp-94 by the His-18 residue, which is absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences have been found in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moment distribution.
Mol Biol (Mosk)
PMID:[Comparison of the heat stability and structure close homologs--Bacillus amyloliquefaciens ribonuclease and Bacillus intermedius 7P ribonuclease]. 848 71

The refolding of ribonuclease T1 involves two major slow processes that exhibit properties of prolyl isomerization reactions. A comparison of the wild-type protein and a designed variant where the cis Ser54-Pro55 bond was replaced by a Gly54-Asn55 bond indicated that the faster of these reactions is the isomerization of Pro55. Here we report the replacement of the other cis proline of ribonuclease T1 at position 39 by alanine. The Pro39Ala variant is similar to the wild-type protein in secondary and tertiary structure, and the enzymatic activity towards RNA and a dinucleotide substrate remains almost unchanged. The fluorescence emission of the single Trp59 is lowered by the Pro39Ala substitution, probably because Trp59 is in close contact to Pro39 in wild-type ribonuclease T1. Unlike the substitution of cis Pro55, the Pro39Ala mutation is strongly destabilizing and reduces the Gibbs free energy of the folded protein by about 20 kJ/mol. Pro39 is buried in native RNase T1 and located near the active site. The observed destabilization could originate from the presence of a cis alanyl bond in the Pro39Ala variant or from a local distortion caused by the incorporation of a trans alanyl peptide bond in the interior of the protein. In the refolding kinetics the replacement of Pro39 leads to a disappearance of the fast-refolding species. Refolding still involves two consecutive slow steps. The first and faster step could be the isomerization of the remaining cis Pro55. The second, very slow step is a novel reaction that appears to have no counterpart in the refolding of the wild-type protein. All mutant molecules must undergo this reaction before reaching the native state. These major changes in the folding kinetics strongly indicate that cis-Pro39 is indeed of major importance for the folding of the wild-type protein. They indicate, moreover, that some new feature of protein folding kinetics is observed in these studies of the Pro39Ala variant.
J Mol Biol 1993 Jun 05
PMID:Stability and folding kinetics of ribonuclease T1 are strongly altered by the replacement of cis-proline 39 with alanine. 851 59

The replacement of cis proline 39 of ribonuclease T1 by an alanine residue leads to a decrease in stability by about 20 kJ/mol and to major changes in the folding kinetics that are not easily explained by the proline model for protein folding. In particular, a novel very slow reaction is observed in the refolding of the Pro39Ala variant. Here the unfolding and refolding kinetics of this protein are further investigated. We show that the very slow reaction is not a prolyl isomerization. It is not created by a slow isomerization of the unfolded protein, nor is it catalyzed by prolyl isomerase, and all molecules have to undergo this reaction during refolding. Most of the unfolded Pro39Ala molecules contain an incorrect trans isomer at the remaining cis Pro55. They use a sequential pathway for refolding, in which trans to cis isomerization at Pro55 precedes the very slow reaction. The refolding of the minor fraction of unfolded Pro39Ala molecules with a correct cis isomer at proline 55 is a single first-order reaction that is limited in rate by the very slow step. The folding mechanism of wild-type ribonuclease T1 cannot be used to explain these results and independent mechanisms are proposed to model the unfolding and refolding of the Pro39Ala variant. The molecular interpretation of the changes in the folding mechanism is tied to the question, as to whether the cis character of the peptide bond at position 38-39 is maintained after the substitution of Pro39 by alanine. A possible explanation could be that the novel very slow folding reaction involves the trans to cis isomerization of the Tyr38-Ala39 bond. Such a reaction is probably slow, since the activation energy is high and since tight coupling with the formation of structure is required to stabilize the cis form of a non-prolyl peptide bond. Alternatively, the strong decrease in folding rate could be correlated with the general destabilization of ribonuclease T1 by the Pro39Ala mutation.
J Mol Biol 1993 Jun 05
PMID:Kinetic models for unfolding and refolding of ribonuclease T1 with substitution of cis-proline 39 by alanine. 851 60

The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.
J Mol Biol 1996 Jan 19
PMID:The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution. 855 22

Initiation of reverse transcription is a crucial step of retroviral infection. In HIV-1, it involves hybridization of the 18 3'-terminal nucleotides of the primer tRNA3(Lys) to the primer binding site (PBS) of the viral RNA. Moreover, additional interactions between the two RNAs were recently evidenced [Isel et al. (1995) J. Mol. Biol. 247, 25269-25272]. To get further information on the topology of the viral RNA/tRNA3(Lys) complex, we used psoralen to induce RNA-RNA crosslinking. A defined intermolecular crosslinked complex was obtained. The crosslinked regions were characterized by RNase T1 digestion followed by bi-dimensional gel electrophoresis. The crosslinked residues (nucleotide mcm5S2U34 and U35 in the anticodon loop of tRNA3(Lys) and UCU154 in the viral RNA upstream of the PBS) were mapped using a retardation method coupled with random hydrolysis. The formation of this crosslink depends on the same elements that are required for the formation of the extended interactions between primer and template RNAs, i.e., the modified bases of the tRNA and a conserved A-rich loop located upstream of the PBS in the genomic RNA. Therefore, the present crosslinking data provide relevant information on the topology of the template/primer binary complex.
 ...
PMID:Psoralen crosslinking between human immunodeficiency virus type 1 RNA and primer tRNA3(Lys). 860 65

Measurements of the change in conformational stability, delta(delta G), upon mutation of two acidic residues at the C terminus of the helix of ribonuclease T1 have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 replaced with Gln, Asp29 replaced with Asn, and the double mutant. In addition, the mutant Lys25 to Gln was studied. Changes in helix content of the peptides with pH confirm the conclusion found in the intact protein, that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in conformational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T1 behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.
J Mol Biol 1996 Nov 01
PMID:The alpha-helix of ribonuclease T1 as an independent stability unit: direct comparison of peptide and protein stability. 891 95


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