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1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis



1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis



Journal of Biomolecular Nmr 7(1): 35-47



The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been investigated by 1H NMR spectroscopy. TOCSY and NOESY experiments in H-2O, tailored to detect both weakly and strongly relaxed resonances, together with steady-state NOEs in both H-2O and D-2O, allowed the identification of 58 of the 59 residues, with one residue near the paramagnetic center undetected. It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cluster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure. Secondary structural elements identified in Tl Fd are a three-stranded antiparallel beta-strand involving the termini of the protein, a double beta-strand (also antiparallel), two alpha-helices and four turns. The existence of a disulfide bridge between the nonligated cysteines is also proposed. Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the 'diamagnetic' protein matrix indicate that the overall folding pattern of Tl Fd is very similar to that of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio gigas (Kissinger et al. (1991) J. Mol. Biol., 219, 693-723). The influence of the paramagnetism of the cluster on the relaxation properties of the protein signals of nonligated residues near the cluster, as well as on the ligated cysteines, correlates well with the proximity to the cluster iron(s), as predicted from the crystal structures for homologous protons of other single-cluster ferredoxins. Finally, the potential role of the various identified structural factors in contributing to the hyperthermostability of this protein is discussed.

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Accession: 008014063

Download citation: RISBibTeXText

PMID: 8720830

DOI: 10.1007/bf00190455


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