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Unfolding Mechanism of Rubredoxin from Pyrococcus furiosus

Cavagnero, Silvia and Zhou, Zhi H. and Adams, Michael W. W. and Chan, Sunney I. (1998) Unfolding Mechanism of Rubredoxin from Pyrococcus furiosus. Biochemistry, 37 (10). pp. 3377-3385. ISSN 0006-2960. doi:10.1021/bi9721804.

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As part of our studies on the structural and dynamic properties of hyperthermostable proteins, we have investigated the unfolding pathways of the small iron−sulfur protein rubredoxin from Pyrococcus furiosus (RdPf) at pH 2. Unfolding has been initiated by temperature jump, triggered by manual mixing of a concentrated protein solution into a thermally preequilibrated buffer. The process has been followed in real time by absorption, tryptophan fluorescence emission, and far-UV circular dichroism. Unlike the case of the mesophilic rubredoxin from Clostridium pasteurianum (RdCp), RdPf displays a complex unfolding kinetics, pointing to the formation of at least three intermediates. All of the steps, including the one involving metal ion release, are extremely slow. However, hydrophobic core relaxation not Fe^(3+) loss is rate-determining for RdPf unfolding. This clearly rules out the fact that Fe^(3+) is solely responsible for the kinetic stability of RdPf. Results have been discussed in terms of sequential vs parallel pathways, and the possible role of irreversible phenomena has been taken into consideration. Aggregation does not appear to play a significant role in the observed kinetic complexities. According to a proposed sequential mechanism, partial release of secondary structure elements precedes iron loss, which is then followed by further loss of β-sheet content and, finally, by hydrophobic relaxation. Although the main features of the RdPf unfolding mechanism remain substantially unchanged over the experimentally accessible temperature range, final hydrophobic relaxation gets faster, relative to the other events, as the temperature is decreased. A qualitative assessment of the unfolding activation parameters suggests that this arises from the very low activation energies (E_a) that characterize this step.

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Chan, Sunney I.0000-0002-5348-2723
Additional Information:© 1998 American Chemical Society. Received September 3, 1997. Publication Date (Web): February 18, 1998. This work was supported by NIH Grant Nos. GM 22432 (S.I.C.) and GM 50736 (M.W.W.A.) from the National Institute of General Medical Sciences, U.S. Public Health Service. We thank Prof. Doug Rees, Dr. Reginald Waldeck, Dr. Brian Schultz and Ms. Lisa Bibbs for interesting and pertinent discussions.
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NIHGM 22432
NIHGM 50736
Issue or Number:10
Record Number:CaltechAUTHORS:20171129-131714433
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Official Citation:Unfolding Mechanism of Rubredoxin from Pyrococcus furiosus. Silvia Cavagnero, Zhi H. Zhou, Michael W. W. Adams, and Sunney I. Chan. Biochemistry 1998 37 (10), 3377-3385. DOI: 10.1021/bi9721804
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:83568
Deposited By: Tony Diaz
Deposited On:29 Nov 2017 23:38
Last Modified:15 Nov 2021 20:12

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