Solution-state structure by NMR of zinc-substituted rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus (original) (raw)
Related papers
PLoS ONE, 2014
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 partiallyunfolded 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.
Modeling the structure of pyrococcus furiosus rubredoxin by homology to other X-ray structures
Protein Science, 2008
The three-dimensional structure of rubredoxin from the hyperthermophilic archaebacterium, Pyrococcus furiosus, has been modeled from the X-ray crystal structures of three homologous proteins from Clostridiumpusteuriunum, Desulfovibrio gigas, and Desulfovibrio vulgaris. All three homology models are similar. When comparing the positions of all heavy atoms and essential hydrogen atoms to the recently solved crystal structure (Day, M.W., et al., 1992, Protein Sci. I, 1494-1507) of the same protein, the homology models differ from the X-ray structure by 2.09 A root mean square (RMS). The X-ray and the zinc-substituted NMR structures (Blake, P.R., et al., 1992b, Protein Sci. I, 1508-1521) show a similar level of difference (2.05 A RMS). On average, the homology models are closer to the X-ray structure than to the NMR structures (2.09 vs. 2.42 A RMS).
Journal of the American Chemical Society, 1998
High-level, all-electron, density functional calculations on a 104-atom model (B3LYP/6-311G** level) have been used, in conjunction with high-resolution X-ray structural data, to predict the remarkable paramagnetic contact shifts recently measured for 1 H, 2 H, 13 C, and 15 N nuclei in Clostridium pasteurianum rubredoxin. Three published X-ray structures for the Fe(III) rubredoxin from C. pasteurianum were employed to construct a 104-atom model for the iron center that included all atoms shown to have strong electronic interactions with the Fe. Each of these models served as a starting point for quantum mechanical calculations at level B3LYP/6-311G**, which, in turn, yielded calculated values for Fermi contact spin densities. The results indicate that the experimental hyperfine shifts are dominated by Fermi contact interactions: calculated Fermi contact spin densities were found to correlate linearly with isotropic hyperfine 1 H, 2 H, 13 C, and 15 N NMR chemical shifts determined for Fe(III) rubredoxin. At the current level of hyperfine peak assignments (all signals assigned to residue and atom types; some assigned to sequence specifically), comparisons were made between experimental shifts and those calculated from the structural model derived from each of the three X-ray structures. For Fe(III) rubredoxin, the R 2 values for the correlation between the calculated spin densities and experimental chemical shifts ranged, depending on the model, from 0.93 to 0.96 for 12 experimental 2 H signals and from 0.85 to 0.96 for 12 experimental 15 N signals. The correlation with experiment was improved by performing partial geometry optimizations at B3LYP/3-21G* on two of the 104-atom models. In particular, the R 2 correlation with experimental 15 N chemical shifts was improved from 0.85 to 0.94 upon optimizing the positions of the nitrogen bound protons reported for one of the X-ray structures. A small increase in the correlation with experiment was also found after optimizing the positions of the Rand -protons of the cysteines of another model. The consistent overall agreement between calculation and experiment supports the validity and usefulness of combining quantum chemical methods with NMR spectroscopy and X-ray crystallography for the testing and refinement of molecular structures. Significantly poorer correlations with experiment were obtained when hypothetical Fe(II) models, derived from two of the X-ray structures of Fe(III) rubredoxin, were used as the basis for the spin density calculations; this suggests that the protein undergoes subtle structural changes upon reduction. † Coordinates for the model structures, the input data for the Gaussian 94 calculations, and the calculated values for the Fermi contact spin densities have been deposited at BioMagResBank (URL: http://www.bmrb.wisc.edu) under the following accession numbers (in parentheses): oxidized rubredoxin models (4118); reduced rubredoxin models (4119). Except as noted above, all calculations were performed at B3LYP/6-311G**.
NMR structure of hypothetical protein TA0938 from Thermoplasma acidophilum
Proteins: Structure, Function, and Bioinformatics, 2007
Introduction. Structural proteomics is an emerging scientific field aiming to obtain one or more representative 3D structures for every structural domain family in nature by application of high throughput structure determination techniques. These structures, and the corresponding protein production vectors and resonance assignments, will provide a valuable resource for structural and functional studies of the thousands of proteins and their homologues that are targeted by the international structural proteomics efforts. 1 T. acidophilum is a thermoacidophilic archaeon 2 that inhabits a hot and highly acidic environment in which few organisms are viable. The genome of T. acidophilum is one of the smallest among free-living organisms. 3 TA0938 is a 110-residue conserved hypothetical protein from T. acidophilum with unknown function. From BLAST 4,5 database search, TA0938 closest homologues are hypothetical proteins in Sulfolobus solfataricus (Q97VD3_SULSO, 5050% sequential identity over 90% of total length) and in Sulfolobus tokodaii (Q96XA7_SULTO, 5036% sequential identity over 86% of total length), both with unknown structure and function. Here, we present the solution structure of TA0938 determined by NMR spectroscopy. On the basis of this solution three-dimensional structure and the amino acid sequence analysis, TA0938 seems to be an uncharacterized protein with a novel fold and a putative Zn-binding motif. These results may be the bases for posterior studies on structure-function relationship in proteins with similar fold.
Biochimie, 2012
Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme e half-life of 1 h at 100 C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 A resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate þ glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous "resilience" of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.