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Distinct Unfolding and Refolding Pathways of Ribonuclease A Revealed by Heating and Cooling Temperature Jumps

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (ΔH#), and activation entropy (ΔS#) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative ΔH# of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro114 isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp115. A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funne

This work was carried out in the frame of COST Chemistry D30-WG005 cooperation and supported by a grant (BFU2006-15543-CO2-02/BMC) from the Ministerio de Educacio´n y Ciencia (Spain)

© Biophysical Journal, 2008, vol. 94, núm. 10, p.4056-4065

Biophysical Society

Autor: Torrent i Mas, Joan
Marchal, Stéphane
Ribó i Panosa, Marc
Vilanova i Brugués, Maria
Georges, Cédric
Dupont, Yves
Lange, Reinhard
Data: 2008
Resum: Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (ΔH#), and activation entropy (ΔS#) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative ΔH# of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro114 isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp115. A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funne
This work was carried out in the frame of COST Chemistry D30-WG005 cooperation and supported by a grant (BFU2006-15543-CO2-02/BMC) from the Ministerio de Educacio´n y Ciencia (Spain)
Format: application/pdf
Cita: Torrent ,J., Marchal, S., Ribó, M., Vilanova, M., Georges, C., Dupont, Y., i Lange, R. (2008). Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps. Biophysical Journal, 94 (10), 4056-4065. Recuperat 14 setembre de 2011, a doi:10.1529/biophysj.107.123893
ISSN: 0006-3495
Accés al document: http://hdl.handle.net/10256/3562
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.107.123893
Articles publicats (D-B)
És part de: © Biophysical Journal, 2008, vol. 94, núm. 10, p.4056-4065
Drets: Tots els drets reservats
Matèria: Ribonucleases
Calor
Fred
Química -- Simulació per ordinador
Chemistry -- Computer simulation
Cold
Heat
Títol: Distinct Unfolding and Refolding Pathways of Ribonuclease A Revealed by Heating and Cooling Temperature Jumps
Tipus: info:eu-repo/semantics/article
Repositori: DUGiDocs

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