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Gorbatyuk, V. Ya.; Chen, Yi Cheng; Wu, Yung Jean; Youle, Richard J.; Huang, Tai Huang

doi:10.1023/a:1008312325225

Cited by 2 publications

(2 citation statements)

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“…15 N HSQC spectrum of (MϪ1, Q1, M23L)rONC has an excellent chemical shift dispersion, all resonances are well resolved, and the complete assignments of backbone and side chain resonances have been reported (24) and deposited in BMRB 4371 and 5835. We used standard two-and three-dimensional heteronuclear NMR techniques to deduce the secondary and tertiary structures.…”

Section: Secondary Structure Determination-thementioning

confidence: 99%

“…The structures of the onconase mutants are also not available. We have used NMR techniques to study the structure-function relationship of the cytotoxic RNases (4,23,24), and the 1 H, 13 C, and 15 N chemical shifts and J HNH␣ coupling constants of (MϪ1, Q1, M23L)rONC have been deposited in BioMagResBank (available on the World Wide Web at www.bmrb.wisc.edu) under BMRB accession numbers 4371 and 5835. In this paper, we describe the solution structure and dynamics of the onconase mutant, (MϪ1, Q1, M23L)rONC, and the dynamics of the E1S-rONC mutant.…”

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confidence: 99%

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Effect of N-terminal and Met23 Mutations on the Structure and Dynamics of Onconase

Gorbatyuk

Tsai

Chang

et al. 2004

Journal of Biological Chemistry

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Onconase (rONC), otherwise known as ranpirnase or P-30 protein, which was initially purified from extracts of Rana pipiens oocytes and early embryos, exhibits anticancer activity both in vitro and in vivo and is in phase III clinical trials for tumor therapy. We have determined the solution NMR structure of a recombinant onconase with Met ؊1 , Gln 1 , and Leu 23 residues (M؊1, Q1, M23L)rONC. The 20 best solution structures had a backbone root mean square deviation of 0.41 ؎ 0.09 Å with respect to the average structure. The energy-minimized average NMR structure had a backbone root mean square deviation of 0.72 Å from the x-ray crystallographic structure of native onconase; however, the orientation of the N-terminal residue in the two structures was very different. Comparison of the 15 N HSQC spectrum of (M؊1, Q1, M23L)rONC with that of a mutant E1S-rONC, which is identical to the nONC except with the N-terminal pyroglutamyl residue replaced by Ser, showed that N-terminal and residue 23 mutations induced structural changes in regions beyond the mutation sites. Model-free analysis of the backbone amide 15 N-T 1 , 15 N-T 2 , and 15 N-1 H NOE relaxation data for (M؊1, Q1, M23L)rONC and E1S-rONC revealed that the E1S-rONC molecule showed very little flexibility, whereas (M؊1, Q1, M23L)rONC exhibited substantial flexibility, which may account for the previously observed reduced stability and increased protease susceptibility. The ␣ 1 helix and ␤-sheets of (M؊1, Q1, M23L)rONC displayed bending motions. These data provided strong evidence for the presence of an N-terminal hydrogen bond network in E1S-rONC, but not in (M؊1, Q1, M23L)rONC.

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Section: Secondary Structure Determination-thementioning

confidence: 99%

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confidence: 99%

Effect of N-terminal and Met23 Mutations on the Structure and Dynamics of Onconase

Gorbatyuk

Tsai

Chang

et al. 2004

Journal of Biological Chemistry

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The Folding Pathway of Onconase Is Directed by a Conserved Intermediate

et al. 2009

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A promising approach to unravel the relationship between sequence information, tertiary structure, and folding mechanism of proteins is the analysis of the folding behavior of proteins with low sequence identity but comparable tertiary structures. Ribonuclease A (RNase A) and its homologues, forming the RNase A superfamily, provide an excellent model system for respective studies. RNase A has been used extensively as a model protein for folding studies. However, little is known about the folding of homologous RNases. Here, we analyze the folding pathway of onconase, a homologous protein from the Northern leopard frog with great potential as a tumor therapeutic, by high-resolution techniques. Although onconase and RNase A significantly differ in the primary structure (28% sequence identity) and in thermodynamic stability (DeltaDeltaG = 20 kJ mol(-1)), both enzymes possess very similar tertiary structures. The present folding studies on onconase by rapid mixing techniques in combination with fluorescence and NMR spectroscopy allow the structural assignment of the three kinetic phases observed in stopped-flow fluorescence spectroscopy. After a slow peptidyl-prolyl cis-to-trans isomerization reaction in the unfolded state, ONC folds via an on-pathway intermediate to the native state. By quenched-flow hydrogen/deuterium exchange experiments coupled with 2D NMR spectroscopy, 31 amino acid residues were identified to be involved in the structure formation of the intermediate. Twelve of these residues are identical in the RNase A sequence, which is a significantly higher percentage (39%) than the overall 28% sequence identity. Moreover, the structure of this intermediate closely resembles two of the intermediates that occur early during the refolding of RNase A. Obviously, in spite of considerable differences in their amino acid sequence the initial folding events of both proteins are comparable, guided by a limited number of conserved residues.

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Cited by 2 publications

References 7 publications

Effect of N-terminal and Met23 Mutations on the Structure and Dynamics of Onconase

Effect of N-terminal and Met23 Mutations on the Structure and Dynamics of Onconase

The Folding Pathway of Onconase Is Directed by a Conserved Intermediate

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