NMR of thermostable proteins

The first NMR structural data on a thermostable Rd was obtained on the Rd from the hyperthermophilic Pyrococcus furiosus, PfR-A, close to 200 °C) 

Ferredoxins (Fds) are widespread in the three domains of life and an abundance of sequence data and structural information are available for Fds isolated from several sources. In particular, the bacterial type Fds are small electron-transfer proteins that posses cubane xFe-yS clusters attached to the protein matrix by Fe ligation of Cys via a conserved consensus ligating sequence. The archaeal type ferredoxins are water-soluble electron acceptors for the acyl-coenzyme A forming 2-oxoacid/ferredoxin oxidoreductase, a key enzyme involved in the central archaeal metabolic pathways. Fds have been distinguished according to the number of iron and inorganic sulphur atoms, 2Fe-2S, 4Fe-4S/3Fe-4S (Fig. Ib-d) and Zn-containing Fds. 

Ferredoxins of the 2Fe-2S type play a role in the photosynthetic electron transport as an essential electron acceptor of photosystem I. The solution 

TlFd (59 amino acids, stable up to 90 °C) contains a single cluster that can exist in both 3Fe-4S and 4Fe-4S forms. The molecular and electronic structures were solved by NMR whereas the X-ray structure is still unknown. Compared to other mesophilic and thermophilic Fds, TTFd showed several structural adjustments such as the addition of a third strand of jS-sheet, a likely Lys2-Glu38 salt bridge from this /1-sheet and the N-terminus and a more hydrophobic and compact interaction between the large /S-sheet and the long helix. According to the authors, each of these modifications contributes to the extraordinary protein thermostability. 

The solution structure of Aspl3Cys, a thermostable mutant of Fd, has been solved by H-NMR and compared to that of the wild-type (WT) protein. The overall folding of the WT protein was maintained in the mutant, except for the immediate vicinity of the new cysteine. The geometry of the new cluster was a typical 4Fe-4S cubane, as monitored by the hyperfine shifts of the co-ordinated cysteines. Conformational heterogeneity, which was partly abolished by heat treatment, was observed and ascribed to a kinetic phenomenon. 

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