Thermodynamic characterization of lysozyme-NAG3 binding in salt-rich mimetic extraterrestrial environments: Implications for protein function

In this study, we investigate the thermodynamic and structural effects of salt-rich mimetic extraterrestrial environments, specifically magnesium perchlorate, magnesium sulfate, magnesium chloride, and sodium chloride, on the binding interaction between hen egg-white lysozyme (Lyz) and N-acetylglucosamine trimer (NAG3), a well-established model for protein–ligand recognition. Using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC), we characterize the binding behavior across a range of temperatures and salt conditions. Our results reveal that Mg(ClO₄)₂ significantly perturbs the binding interaction, reducing the binding constant by an order of magnitude and altering both enthalpic and entropic contributions. CD spectroscopy reveals subtle perturbations in lysozyme's tertiary structure, while DSC analysis indicates a decrease in both denaturation temperature and enthalpy, consistent with a modified hydration shell. These findings demonstrate that salts-rich environments can modulate protein–ligand interactions primarily through hydration and solvation effects, without necessarily compromising structural integrity. This work offers critical insights into the biochemical resilience of proteins in salt-rich environments, offering meaningful implications for the ongoing search for life in extraterrestrial environments.