Structural determinants responsible for the thermostability of Sso7d and its single point mutants

Abstract

Sso7d is a small protein especially attractive as a model for characterizing by NMR, the structural determinants responsible for thermal stability. With this aim, structural parameters of the Sso7d protein have been compared with those of single point mutants, in particular with K12L, more stable (T m around 1008C) and F31A, less stable (T m 618C) than the wild-type. In addition, a mutant lacking eight residues of the C-terminal helix (T m 508C) has been studied to investigate the role of the helix in structure stabilization. Melting temperatures, DDG's of unfolding, cavities, electrostatic interactions, solvent accessible areas, hydrophobic cores, and aromatic cluster geometries have been analyzed in order to gain insights into the key structural factors determining the thermostability differences. Our data suggest that absence of cavities and a larger salt bridge network represent the major determinants contributing to the enhanced stability of K12L relative to the Sso7d protein and its mutant F31A.Introduction. The interest for thermophilic proteins has increased during the last years for several reasons: first of all they can open a wide field of microbiological and technical industrial applications and processes, furthermore, they provide excellent models for understanding the molecular mechanisms underlying protein folding and stabilization, and for describing their functional properties. The stability of proteins from organisms living under extreme conditions is a long standing issue and different authors attributed several factors affecting their greater structural stability with respect to the mesophilic counterpart. 1 It has been pointed out that there is not a unique structural requirement for making a protein thermostable. Several factors have been found to play a relevant role in the protein stability, such as increased hydrophobic and aromatic contacts, optimization of charge-charge interactions, and side chain packing 1 ; however, the mechanisms by which these proteins attain thermostability remain unclear.In the last years we have studied the recombinant form of Sso7d, initially isolated from the thermoacidophilic archeaobacterium Sulfolobus solfataricus living in volcanic hot springs at 878C and displaying a pressure resistance up to 14 kbar. 2 The protein is composed of 62

Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability

“…The drop in activities could be due to conformational changes induced by mutations, which could alter binding. Indeed, even a single mutation introduced in WT Sso7d was reported to lead to a reorientation of neighboring lateral chains while preserving the overall fold of the protein (Consonni et al, 2007). Furthermore, we have observed with the three-dimensional structure of the C3 anti-IgG Affitin that, while the fold of Sac7d was maintained upon mutagenesis necessary for its generation, a significant backbone deviation occurred in a ␤-turn-␤ region with respect to the crystal structures of WT Sac7d (Béhar et al, 2013).…”

“…This was not observed as the T m of D1Sso7d was decreased by 6.5 • C, indicating that some determinants for the higher thermal stability of Sso7d were altered by the grafting of the hIgG binding site. Similarly, the single mutations K13L and E36L identified as thermally stabilizing WT Sso7d (Consonni et al, 2007) did not stabilize D1Sso7d. However, the combination of both mutations was able to produce a stabilization effect (+2.7 • C) and the final D1Sso7d-DM was nearly as stable as the original D1Sac7d (76.9 • C vs. 80.7 • C).…”

“…1C and D). Two single mutants of D1Sso7d (K13L and E36L) were constructed, as previous studies with wild type Sso7d have described their role in thermal stabilization (Consonni et al, 2007). The double mutant K13L and E36L of D1Sso7d, hereafter called D1Sso7d-DM, was also produced to investigate a potential additive effect on stability.…”

“…To this end, we grafted the binding site of the Affitin D1 derived from Sac7d, specific for human IgG and with a thermal stability of 80.7 • C (Béhar et al, 2013), into the thermally more stable Sso7d protein. We also produced this chimera with two single mutations, described as thermally stabilizing wild type Sso7d (Consonni et al, 2007), and with the corresponding double mutation. We then studied the function and the thermal and pH stability properties of the resulting proteins.…”

Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

“…31,43 Mutagenesis of residues in the hydrophobic core results in a significant decrease in the melting temperature of Sso7d. 44 In particular, the single point mutation F31A was shown to decrease the melting temperature by 24°C. 45 Similarly, deletion of the C-terminal α helix causes a 46°C decrease in melting temperature.…”