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Hyperthermophile Protein Behavior: Partially-Structured Conformations of Pyrococcus furiosus Rubredoxin Monomers Generated through Forced Cold-Denaturation and Refolding
- Source :
- PLoS ONE, PLoS ONE, Vol 9, Iss 3, p e80014 (2014)
- Publication Year :
- 2014
- Publisher :
- Public Library of Science, 2014.
-
Abstract
- Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as ‘Trishanku’ unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native ‘Trishanku’ refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.
- Subjects :
- Models, Molecular
Circular dichroism
Protein Folding
Protein Denaturation
Hot Temperature
Protein Conformation
Archaeal Proteins
Molecular Sequence Data
Biophysics
lcsh:Medicine
Redox
Biochemistry
Protein Refolding
Protein structure
Rubredoxin
Denaturation (biochemistry)
Amino Acid Sequence
Biomacromolecule-Ligand Interactions
lcsh:Science
Biology
Bioinorganic Chemistry
Multidisciplinary
biology
Base Sequence
Chemistry
Protein Stability
Physics
Circular Dichroism
Rubredoxins
lcsh:R
Proteins
Sequence Analysis, DNA
biology.organism_classification
Hyperthermophile
Cold Temperature
Pyrococcus furiosus
Crystallography
Protein Classes
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Thermodynamics
lcsh:Q
Electrophoresis, Polyacrylamide Gel
Oxidation-Reduction
Cysteine
Research Article
Subjects
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 9
- Issue :
- 3
- Database :
- OpenAIRE
- Journal :
- PLoS ONE
- Accession number :
- edsair.doi.dedup.....0069d449f4d45db1d36b70fc27981a67