Erlet Shehi

Exploring hyperthermophilic proteins under pressure: theoretical aspects and experimental findings

Proteins from hyperthermophilic microorganisms are generally capable of withstanding temperatures close to, or even higher than the boiling point. As a rule, these proteins are strongly piezostable as well, although exceptions have been also reported. This observation has a theoretical relevance, as the understanding of the effects of pressure and temperature on protein stability is equally important to develop a comprehensive model for their thermodynamic stability. Nevertheless, the structural features justifying the correlation between heat resistance and pressure resistance are poorly understood. Actually, most reports do not exceed the phenomenological level. Only in the case of the small protein Sso7d from Sulfolobus solfataricus, characterisation of wild-type and some mutants showed that both properties are largely accounted for by a network of aromatic residues found in the hydrophobic core of the molecule. Current knowledge, however, does not allow to establish to what extent this finding may be generalised. In a biotechnological perspective, hyperthermophilic enzymes seem to be more suitable for bioprocesses at high pressure with respect to their mesophilic counterparts. Indeed, thanks to their higher resistance towards pressure and temperature, they may be exploited in a much broader range of working conditions for tuning activity and specificity. Furthermore, they are often activated by increasing pressure, although it cannot be established, to date, to what extent this is a common feature.

The Sso7d DNA‐binding protein from Sulfolobus solfataricus has ribonuclease activity

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double‐stranded DNA without sequence specificity by placing its triple‐stranded β‐sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single‐point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double‐ and single‐stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.

Hydrostatic Pressure Effects on the Stability of Ataxin-3

Protein misfolding and formation of amyloids are the major event in the development of neurodegenerative diseases but the mechanism of this biological phenomenon remains to be elucidated. Here, we report the high pressure denaturation of the spinocerebellar ataxia type 3 protein Ataxin-3 from man and mouse using fluorescence spectroscopy. The two proteins carry 26 and 6 consecutive glutamines, respectively. The tryptophan fluorescence measurements indicated that at pH 7.5 and 25°C the pressure denaturation is reversible but with a large hysteresis in the renaturation profile for both proteins. The reversibility of the pressure-induced unfolding was confirmed by the study of 8-anilinonaphtalene-1-sulfonate (ANS) binding to both proteins. Interestingly, thioflavin-T binding is observed at high pressure, revealing a conversion of the native Ataxin-3 into a potential amyloidogenic state for both proteins. These data provide strong evidence that studying protein folding by hydrostatic pressure may contribute to a better understanding of the mechanism of protein misfolding leading to polyglutamine neurodegenerative diseases.

Structural prerequisites for the stability of Sso7d from the archaeon Sulfolobus solfataricus versus high pressure and temperature

Abstract Sso7d is a protein purified from the thermoaddophilic archaeon Sulfolobus solfataricus. In this paper we present the effect of pressure and temperature on the stability of Sso7d and its mutants. Fourth derivative UV spectroscopy and ANS (I-anilinonaphtalene-8-sulfonate) binding were used to monitor the protein unfolding.