Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima

Abstract

Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities. Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems. Small single-domain proteins are suitable candidates to overcome these obstacles. Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry. The unfolding transition can be described over a broad pH range (3.5–8.5) by a reversible two-state process. Maximum stability (ΔG (25 °C) = 6.5 kcal/mol) was observed at pH 5–6 where Tm Csp unfolds with a melting temperature at 95 °C. The heat capacity difference between the native and the denatured states is 1.1(±0.1) kcal/(mol K). At pH 7, thermal denaturation occurs at 82 °C. The corresponding free energy profile has its maximum at 30 °C with ΔGN → U = 4.8(±0.5) kcal/mol. At the optimal growth temperature of T. maritima (80 °C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy. With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules.