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Destabilizing Mutations Alter the Hydrogen Exchange Mechanism in Ribonuclease A

The 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 (35ºC), the I106A variant (35ºC), and the V108G variant (10ºC) 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 by 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 1 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

© Biophysical Journal, vol. 94, núm. 6, p. 2297–2305

Biophysical Society

Autor: Bruix, Marta
Ribó i Panosa, Marc
Benito i Mundet, Antoni
Data: març 2008
Resum: The 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 (35ºC), the I106A variant (35ºC), and the V108G variant (10ºC) 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 by 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 1 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
Format: application/pdf
Cita: Bruix, M., Ribó, M., Benito, A., Laurents,D.V., Rico, M., i Vilanova, M. (2008). Destabilizing Mutations Alter the Hydrogen Exchange Mechanism in Ribonuclease A. Biophysical Journal, 94 (6), 2297–2305. Recuperat 12 juliol de 2011,a http://www.cell.com/biophysj/archive/issue?pii=S0006-3495%2808%29X7013-4
ISSN: 0006-3495
Accés al document: http://hdl.handle.net/10256/3483
Llenguatge: eng
Editor: Biophysical Society
Col·lecció: MEC/PN 2006-2009/BFU2006-15543-CO2-02
Reproducció digital del document publicat a: http://dx.doi.org/10.1529/biophysj.107.122952
Articles publicats (D-B)
És part de: © Biophysical Journal, vol. 94, núm. 6, p. 2297–2305
Drets: Tots els drets reservats
Matèria: Ribonucleases
Títol: Destabilizing Mutations Alter the Hydrogen Exchange Mechanism in Ribonuclease A
Tipus: info:eu-repo/semantics/article
Repositori: DUGiDocs

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