Comparison of the primary structures of ribonuclease U2 isoforms

Kanaya, Shigenori; Uchida, Tsuneko

doi:10.1042/bj2400163

Cited by 26 publications

(17 citation statements)

References 19 publications

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“…transferase 1BZY (48,49), human insulin 2HIU (NMR) (50,51), mouse interleukin 1␤ 2MIB (52,53), human interleukin 2 3INK (54, 55), chicken lysozyme 1E8L (NMR) (56,57), bovine ribonuclease A 1AFK (58,59), Ustilago sphaerogena ribonuclease U2 1RTU (60,61), bovine seminal ribonuclease 11BG (62,63), human T cell surface protein CD4 1CDJ (64,65), human thioltransferase 1JHB (NMR) (66,67), human triosephosphate isomerase 1HTI (68,69), and bovine trypsin 1MTW (70,71).…”

Section: Methodsmentioning

confidence: 99%

Prediction of protein deamidation rates from primary and three-dimensional structure

Robinson

2001

Proc. Natl. Acad. Sci. U.S.A.

232

247

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A method for the quantitative estimation of instability with respect to deamidation of the asparaginyl (Asn) residues in proteins is described. The procedure involves the observation of several simple aspects of the three-dimensional environment of each Asn residue in the protein and a calculation that includes these observations, the primary amino acid residue sequence, and the previously reported complete set of sequence-dependent rates of deamidation for Asn pentapeptides. This method is demonstrated and evaluated for 23 proteins in which 31 unstable and 167 stable Asn residues have been reported and for 7 unstable and 63 stable Asn residues that have been reported in 61 human hemoglobin variants. The relative importance of primary structure and threedimensional structure in Asn deamidation is estimated.biological clocks ͉ proteins T he spontaneous deamidation of glutaminyl and asparaginyl residues causes experimentally and biologically important changes in peptide and protein structures. In asparaginyl deamidation, the primary reaction products are aspartyl and isoaspartyl. Early work on peptide and protein deamidation (1-10) established that deamidation occurs in vitro and in vivo and depends on primary sequence, three-dimensional (3D) structure, pH, temperature, ionic strength, buffer ions, and other solution properties.It was hypothesized (3, 5, 7) and then experimentally demonstrated (2, 8, 9, 11) that deamidation can serve as a biologically relevant molecular clock that regulates the timing of in vivo processes. Substantial evidence supports the hypothesis that Asn deamidation at neutral pH proceeds through a cyclic imide reaction mechanism (12)(13)(14).A procedure is needed whereby the stability of individual amides in peptides and proteins can be reliably estimated. Although it was evident to investigators 30 years ago (2-7) that protein deamidation rates depend on primary, secondary, tertiary, and quaternary protein structure, and numerous examples have been found, it was not possible to devise a useful deamidation prediction procedure until a complete library of deamidation rates as a function of primary sequence was available.A suitable library of sequence-determined Asn rates has now been published (15), and the relevance of this library has been established (16). These rates can now be combined with 3D data to provide a useful deamidation prediction procedure. Each amide residue has an intrinsic sequence-determined deamidation rate, which depends on charge distribution, steric factors, and other aspects of peptide chemistry. This primary rate is modulated by 3D structure, which usually slows the rate. In a few instances, it increases the deamidation rate.We have devised a simple procedure that is useful for predicting the relative deamidation rates of most protein Asn residues. We have tested this procedure on a complete set of all proteins for which, during a review of the literature, we found experiments specifically identifying one or more labile Asn residues in a protein and also a suitable 3...

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Section: Methodsmentioning

confidence: 99%

Prediction of protein deamidation rates from primary and three-dimensional structure

Robinson

2001

Proc. Natl. Acad. Sci. U.S.A.

232

247

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“…The presence of isoaspartate, in which the former side chain is now part of the peptide backbone, can functionally impair peptides and proteins (Graf et al, 1973;Kanaya & Uchida, 1986;Johnson et al, 1987a;Hallahan et al, 1992). ProIonged methylation by PIMT in vitro has been shown to convert isoaspartyl peptides to the aspartyl form (Johnson et al, 1987b;McFadden & Clarke, 1987;Galletti et al, 1988a) and to partially restore functionality of deamidated CaM (Johnson et al, 1987a).…”

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confidence: 99%

In vitro aging of calmodulin generates isoaspartate at multiple Asn–Gly and Asp–Gly sites in calcium‐binding domains II, III, and IV

Potter

Henzel²,

Aswad

1993

Protein Science

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We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyL3H from S-adenosyl-~-[methyl-~H]methionine to the isoaspartyl (a-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 "C. Analysis of proteolytic peptides of aged calmodulin revealed that >95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains 111, IV, and I1 accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain 111) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain 11. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain 11, and to Asp-93 and/or Asp-95 in domain 111. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.

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“…For example, one of its most distinctive features is the absence of any Lys residue and the presence of just a single Arg, what results in a highly acidic pI value of about 3.0 [1,17,18], and an anomalous electrophoretic behavior, even in the presence of SDS [19]. It is also a peculiar protein from a spectroscopic point of view since it contains a single but strongly quenched Trp residue [20,21].…”

Section: Introductionmentioning

confidence: 99%

“…It is also a peculiar protein from a spectroscopic point of view since it contains a single but strongly quenched Trp residue [20,21]. Natural wild-type RNase U2 in solution is slowly converted upon ageing into two other isoforms containing one or two isoaspartate bonds 4 and different secondary structure and specific enzymatic activity [17,[21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris

Álvarez-García

Garcı́a-Ortega

Rı́os

et al. 2009

Protein Expression and Purification

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Ribonuclease U2, secreted by the smut fungus Ustilago sphaerogena, is a cyclizing ribonuclease that displays a rather unusual specificity within the group of microbial extracellular RNases, best represented by RNase T1.Superposition of the three-dimensional structures of RNases T1 and U2suggests that the RNase U2 His 101 would be the residue equivalent to the RNase T1 catalytically essential His 92. RNase U2 contains three disulfide bridges but only two of them are conserved among the family of fungal extracellular RNases. The non-conserved disulfide bond is established between Cys residues 1 and 54. Mispairing of the disulfide network due to the presence of two consecutive Cys residues (54 and 55) has been invoked to explain the presence of wrongly folded RNase U2 species when produced in P. pastoris. In order to study both hypotheses, the RNase U2 H101Q and C1/54S variants have been produced, purified, and characterized. The results obtained support the major conclusion that His 101 is required for proper protein folding when secreted by the yeast P. pastoris. On the other hand, substitution of the first Cys residue for Ser results in a mutant version which is more efficiently processed in terms of a more complete removal of the yeast α-factor signal peptide. In addition, it has been shown that elimination of the Cys 1-Cys 54 disulfide bridge does not interfere with RNase U2 proper folding, generating a natively folded but much less stable protein.

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Comparison of the primary structures of ribonuclease U2 isoforms

Cited by 26 publications

References 19 publications

Prediction of protein deamidation rates from primary and three-dimensional structure

Prediction of protein deamidation rates from primary and three-dimensional structure

In vitro aging of calmodulin generates isoaspartate at multiple Asn–Gly and Asp–Gly sites in calcium‐binding domains II, III, and IV

Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris

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