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Destabilizing mutations alter the hydrogen exchange mechanism in Ribonuclease A

AuthorsBruix, M. CSIC ORCID; Ribó, M.; Benito, A.; Laurents, D.V. CSIC ORCID; Rico, Manuel CSIC; Vilanova, M.
Issue Date15-Mar-2008
PublisherBiophysical Society
CitationBiophysical Journal 94: 2297-2305 (2008)
AbstractThe effect of strongly destabilizing mutations, I106A and V108G of Ribonuclease A (RNase A), on its structure and stability has been determined by NMR. The solution structures of these variants are essentially equivalent to RNase A. The exchange rates of the most protected amide protons in RNase A (35°C), the I106A variant (35°C), and the V108G variant (10°C) yield stability values of 9.9, 6.0, and 6.8 kcal/mol, respectively, when analyzed assuming an EX2 exchange mechanism. Thus, the destabilization induced b y these mutations is propagated throughout the protein. Simulation of RNase A hydrogen exchange indicates that the most protected protons in RNase A and the V108G variant exchange via the EX2 regime, whereas those of I106A exchange through a mixed EX1 + EX2 process. It is striking that a single point mutation can alter the overall exchange mechanism. Thus, destabilizing mutations joins high temperatures, high pH and the presence of denaturating agents as a factor that induces EX1 exchange in proteins. The calculations also indicate a shift from the EX2 to the EX1 mechanism for less protected groups within the same protein. This should be borne in mind when interpreting exchange data as a measure of local stability in less protected regions. © 2008 by the Biophysical Society.
Description9 pags, 6 figs
Publisher version (URL)http://dx.doi.org/10.1529/biophysj.107.122952
Identifiersdoi: 10.1529/biophysj.107.122952
issn: 0006-3495
Appears in Collections:(IQFR) Artículos
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