Item


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

Author: Bruix, Marta
Ribó i Panosa, Marc
Benito i Mundet, Antoni
Date: 2008 March
Abstract: 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
Citation: 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
Document access: http://hdl.handle.net/10256/3483
Language: eng
Publisher: Biophysical Society
Collection: 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)
Is part of: © Biophysical Journal, vol. 94, núm. 6, p. 2297–2305
Rights: Tots els drets reservats
Subject: Ribonucleases
Title: Destabilizing Mutations Alter the Hydrogen Exchange Mechanism in Ribonuclease A
Type: info:eu-repo/semantics/article
Repository: DUGiDocs

Subjects

Authors


Warning: Unknown: write failed: No space left on device (28) in Unknown on line 0

Warning: Unknown: Failed to write session data (files). Please verify that the current setting of session.save_path is correct (/var/lib/php5) in Unknown on line 0