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^,

Abstract

Sso7d is a basic 7-kDa DNA-binding protein from Sulfolobus solfataricus, also endowed with ribonuclease activity. The protein consists of a double-stranded antiparallel beta-sheet, onto which an orthogonal triple-stranded antiparallel beta-sheet is packed, and of a small helical stretch at the C-terminus. Furthermore, the two beta-sheets enclose an aromatic cluster displaying a fishbone geometry. We previously cloned the Sso7d-encoding gene, expressed it in Escherichia coli, and produced several single-point mutants, either of residues located in the hydrophobic core or of Trp23, which is exposed 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 structure 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 finding is the change in orientation of the Trp23 side chain, which in the wild type is completely exposed to the solvent, whereas in the mutant is largely 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 mutation, notably a strong distortion in the beta-sheets with loss in several hydrogen bonds, increased flexibility of some stretches of the backbone, and some local strains. On one hand, these features may justify the dramatic destabilization 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 result of a single-point mutation.

Crystal Structure of a Thermophilic Cytochrome P450 from the Archaeon Sulfolobus solfataricus

“…. led to an unprecedented loss" of thermal-and baro-stability by at least 27 K and 10 kilobar, respectively (36). Future mutagenesis work on CYP119 should reveal if the aromatic clustering revealed by the CYP119 crystal structure is the key to its increased thermal stability.…”

Two Amino Acid Substitutions within the First External Loop of CCR5 Induce Human Immunodeficiency Virus-Blocking Antibodies in Mice and Chickens

“…The volume of the assigned NOE cross-peaks from the NOESY spectra was measured by using the standard Felix integration routines and was converted into distances (1). Parameters used for structure calculations were described elsewhere (15).…”

Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

“…According to the thermodynamic and structural investigations on F31A-Sso7d [17][18][19][20], and the kinetic measurements on several mutant forms of Sso7d by Guerois and Serrano [47], it emerges that the residues constituting the hydrophobic core of the protein play a crucial role in order to render the native structure extra-stable against temperature, pressure and GuHCl. This does not contrast with the current idea that favourable electrostatic interactions among charged groups placed on the protein surface are the specific tool to cope with high temperature [48][49][50]; see also the percentage values of the ratio of the number of charged residues over the total for the SH3-like proteins listed in Table 3.…”

“…The recombinant protein proved to be indistinguishable from its natural counterpart on the basis of its DNA-binding and ribonuclease activities and stability against temperature, even though no lysine monomethylation was found [20]. The purity of the protein was confirmed by means of SDS-PAGE and MALDI-TOF mass spectrometry.…”

“…There are no asparagines, histidines and cysteines. The thermal stability of Sso7d has been deeply investigated, showing that the protein has a denaturation temperature close to the boiling point of water [16][17][18][19][20]. However, the stability of Sso7d against the denaturing action of GuHCl has not been characterized.…”