A single-point mutation in the extreme heat- and pressure-resistant Sso7d protein from Sulfolobus solfataricus leads to a major rearrangement of the hydrophobic core

Sso7d is a basic 7-kDa DNA-binding protein from Sulfolobus solfataricus, al so endowed with ribonuclease activity. The protein consists of a double-str anded antiparallel beta-sheet, onto which an orthogonal triple-stranded ant iparallel beta-sheet is packed, and of a small helical stretch at the C-ter minus. Furthermore, the two beta-sheets enclose an aromatic cluster display ing a fishbone geometry. We previously cloned the Sso7d-encoding gene, expr essed it in Escherichia coli, and produced several single-point mutants, ei ther of residues located in the hydrophobic con or of Trp23, which is expos ed to the solvent and plays a major role in DNA binding. The mutation F31A was dramatically destabilizing, with a loss in thermo-and piezostabilities by at least 27 K and 10 kbar, respectively. Here, we report the solution st ructure of the F31A mutant, which was determined by NMR spectroscopy using 744 distance constraints obtained from analysis of multidimensional spectra in conjunction with simulated annealing protocols. The most remarkable fin ding is the change in orientation of the Trp23 side chain, which in the wil d type is completely exposed to the solvent, whereas in the mutant is large ly buried in the aromatic cluster. This prevents the formation of a cavity in the hydrophobic core of the mutant, which would arise in the absence of structural rearrangements. We found additional changes produced by the muta tion, notably a strong distortion in the beta-sheets with loss in several h ydrogen bonds, increased flexibility of some stretches of the backbone, and some local strains. On one hand, these features may justify the dramatic d estabilization provoked by the mutation; on the other hand, they highlight the crucial role of the hydrophobic core in protein stability. To the best of our knowledge, no similar rearrangement has been so far described as a r esult of a single-point mutation.