Contribution of structural peculiarities of onconase to its high stability and folding kinetics.


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. Study holds ProTherm entries: 19905, 19906, 19907, 19908, 19909, 19910, 19911, 19912, 19913, 19914 Extra Details: hydrophobic cluster; disulfide bond; unfolding kinetics

Submission Details


Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Arnold U;Schulenburg C;Schmidt D;Ulbrich-Hofmann R,Biochemistry (2006) Contribution of structural peculiarities of onconase to its high stability and folding kinetics. PMID:16533040
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1PU3 2003-06-24T00:00:00+0000 0 The Solution NMR Structure and Dynamics of a Recombinant Onconase with Altered N-terminal and Met23 residues
1YV4 2005-02-15T00:00:00+0000 1.51 X-ray structure of M23L onconase at 100K
1YV6 2005-02-15T00:00:00+0000 1.78 X-ray structure of M23L onconase at 298K
1YV7 2005-02-15T00:00:00+0000 1.9 X-ray structure of (C87S,des103-104) onconase
2GMK 2006-04-06T00:00:00+0000 1.65 Crystal structure of onconase double mutant with spontaneously-assembled (AMP) 4 stack
2I5S 2006-08-25T00:00:00+0000 1.9 Crystal structure of onconase with bound nucleic acid
2KB6 2008-11-21T00:00:00+0000 0 Solution structure of onconase C87A/C104A
2LT5 2012-05-14T00:00:00+0000 0 Zymogen-FLG of the onconase
3FD7 2008-11-25T00:00:00+0000 1.53 Crystal structure of Onconase C87A/C104A-ONC

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Protein P-30 P22069 RNP30_LITPI