| Contribution of structural peculiarities of onconase to its high stability and folding kinetics. | |
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MedLine Citation:
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PMID: 16533040 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Onconase (ONC) from Rana pipiens is the smallest member of the ribonuclease A (RNase A) superfamily. Despite a tertiary structure similar to RNase A, ONC is distinguished by an extremely high thermodynamic stability. In the present paper we have probed the significance of three structural regions, which exhibit structural peculiarities in comparison to RNase A, for the stability of ONC to temperature and guanidine hydrochloride induced denaturation: (i) the N-terminal pyroglutamate residue, (ii) the hydrophobic cluster between helix I and the first beta-sheet, and (iii) the C-terminal disulfide bond. For this purpose, the enzyme variants <E1E-, <E1P-, F28T-, F28A-, F36Y-, and C87A/C104A-ONC were produced and studied in equilibrium and kinetic measurements. The destabilizing influence of the mutations strongly depended on the modified structural region. The exchanges of the N-terminal pyroglutamate (<E1E- and <E1P-ONC) had the smallest impact (DeltaDeltaG([D])50% = 4.2 and 7.0 kJ mol(-)(1)), while interferences in the hydrophobic cluster (F28T-, F28A-, and F36Y-ONC) had larger effects (DeltaDeltaG([D])50% = 22.2, 20.9, and 19.5 kJ mol(-)(1)). The removal of the C-terminal disulfide bond (C87A/C104A-ONC) showed the largest influence on stability (DeltaDeltaG([D])50% = 32.0 kJ mol(-)(1)). As concluded from the comparison of DeltaDeltaG([D])50% and DeltaDeltaG++(U)[D]50%, all destabilization effects were exclusively caused by increased unfolding rate constants except for C87A/C104A-ONC, where unfolding as well as folding was impacted. Of all amino acid residues investigated, Phe28, which is unique for ONC among the ribonucleases, had the greatest importance for rate of unfolding. Our data on the folding and unfolding kinetics indicate that the strong stabilization of ONC in comparison to RNase A is caused by a dramatic deceleration of the unfolding reaction. |
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Authors:
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Ulrich Arnold; Cindy Schulenburg; Doreen Schmidt; Renate Ulbrich-Hofmann |
Publication Detail:
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Type: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Biochemistry Volume: 45 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 2006 Mar |
Date Detail:
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Created Date: 2006-03-14 Completed Date: 2006-05-16 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 3580-7 Citation Subset: IM |
Affiliation:
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Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes Strasse 3, 06120 Halle, Germany. ulrich.arnold@biochemtech.uni-halle.de |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Substitution
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physiology Animals Circular Dichroism Enzyme Stability / physiology* Guanidine / metabolism, pharmacology Hydrophobicity Kinetics Protein Denaturation Protein Folding* Protein Renaturation Protein Synthesis Inhibitors / metabolism* Rana pipiens / metabolism Ribonuclease, Pancreatic / chemistry, metabolism Ribonucleases / genetics, metabolism* Temperature Thermodynamics |
| Chemical | |
Reg. No./Substance:
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0/Protein Synthesis Inhibitors; 113-00-8/Guanidine; EC 3.1.-/Ribonucleases; EC 3.1.-/ranpirnase; EC 3.1.27.5/Ribonuclease, Pancreatic |
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