Comparing the thermal, chemical and mechanical stabilities of extremophilic cold shock protein variants

Proteins conduct a vast array of chemical processes and achieve this through a balance of flexibility and function. Proteins from extremophilic organisms have evolved to adjust this balance to function in extreme conditions. Research into hot-adapted proteins has been encouraged through successes in improving enzyme thermostability, though cold-adapted proteins also offer substantial potential as they can function effectively at lower temperatures. Research into cold-adapted proteins remains limited despite cold climates comprising over 80% of Earth’s biosphere.

This study compares the stabilities of cold shock protein (Csp) homologues from bacteria growing at vastly different temperatures. The cold-adapted Csps show reduced thermal denaturation mid-points (Tm values) compared to a temperate Csp with PB6 Csp displaying the lowest Tm of 42.8 ⁰C contrasting with 52.3 ⁰C shown by BsCsp. Thermostability projections using the Gibbs-Helmholtz equation show cold-adapted Csps display slightly reduced thermostabilities and remain folded over a narrower range of temperatures. Csp thermostabilities were found to be very similar at around 8 degrees below the optimal growth temperature of the bacteria each Csp was derived from, suggesting thermostabilities evolved relating to Csp operating temperatures. The roles of electrostatics and hydrophobic packing in stabilizing a hyperthermophilic Csp are evaluated using mutants that highlight how a single side chain length reduction dramatically decreases Csp thermostability. Kinetic studies showed that the Csps exhibit similar folding rate constants but different unfolding rate constants. Initial flexibility studies suggest that DNA binding increases Csp rigidity and triggers a conformational change in a psychrotrophic Csp.

Csp mechanical stabilities showed the same hierarchy as thermostabilities and similar sensitivity to temperature changes. Energy landscape projections constructed using Monte Carlo simulations showed the mechanical energy barrier height of Csp unfolding was temperature independent. At 5 ⁰C the Csps showed similar mechanical softness however the hyperthermophilic TmCsp shows greater mechanical softness at higher temperatures.