title: Unfolding mechanism of rubredoxin from Pyrococcus furiosus.
abstract: 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 Fe3+ loss--is rate-determining for RdPf unfolding. This clearly rules out the fact that Fe3+ 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 beta-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 (Ea) that characterize this step.
authors.affiliation: Arthur Amos Noyes Laboratories of Chemical Physics, California Institute of Technology, Pasadena 91125, USA.
authors.forename: S
authors.initials: S
authors.lastname: Cavagnero
authors.forename: Z H
authors.initials: ZH
authors.lastname: Zhou
authors.forename: M W
authors.initials: MW
authors.lastname: Adams
authors.forename: S I
authors.initials: SI
authors.lastname: Chan
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002942:Circular Dichroism; D003013:Clostridium / Q000737:chemistry; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D019711:Pyrococcus / Q000737:chemistry*; D012416:Rubredoxins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D012416:Rubredoxins