Addition of subunit γ, K+ ions, and lipid restores the thermal stability of solubilized Na,K-ATPase

► Thermal unfolding of the active site region of Na,K-ATPase may occur at 55 °C. ► Transition at 48 °C in thermal unfolding profile is dependent on subunit γ. ► E1 conformation is more labile towards thermal denaturation than E2. ► Lipid bilayer has a larger protection against thermal unfolding vs. detergent micelle. ► Closed and open conformations have different thermal unfolding profiles.