Using Single Molecule Force Spectroscopy to Probe Proteins from Extremophiles

Abstract

Extremophiles are organisms which survive and thrive in the most extreme chemical and physical conditions on Earth. The proteins from extremophilic organisms play a key role in enabling them to survive and function in specific environmental extremes. These proteins are of great interest as they have the ability to retain their folded structure and to possess the necessary flexibility to be functional under conditions which normally denature proteins. For this reason, they offer attractive, model systems in which to explore the origin of protein structure and dynamics under different, extreme environmental conditions. We use single molecule force spectroscopy to measure the mechanical stability and flexibility of proteins derived from extremophile organisms. We have characterised the mechanical stability of a cold shock protein from the hypethermophilic organism, Thermotoga maritima in the temperature range 5-40°C. We measure temperature-dependent changes in features of the unfolding energy landscape of this protein by studying the pulling speed dependence of the unfolding force with temperature in combination with Monte Carlo simulations. We find that the position of the transition state to unfolding shifts away from the native state with increased temperature, reflecting a reduction in the spring constant of the protein and an increase in the malleability of the structure. The mechanical robustness and malleability of this cold shock protein over the temperature range studied, provides an insight into the dynamical properties of hyperthermophilic proteins. To gain further insight into the kinetic stability and adaptation strategies of this protein we are examining structural homologues from mesophilic organisms and mutants of the cold shock protein. These will provide a deeper understanding of the adaptations found in hyperthermophilic proteins, and will enable the rational design of proteins for bionanotechnological applications.