Determining Factors for Spermidine-Induced Modulation of Conformational Stability and Dynamics of Horse Myoglobin

The present work describes how water-miscible natural polyamine (NPA) spermidine (SPD) alters the conformational stability and dynamics of horse myoglobin (h-Mb) at pH 7.4. Analysis of thermal- and chemical-induced unfolding profiles of h-Mb at different concentrations of SPD at pH 7.4 revealed that SPD (≥5 mM) reduces the conformational stability of h-Mb. The effect of SPD was further corroborated using MD simulations, which showed that SPD (≥5 mM) enhanced the conformational fluctuations and reduced the structural stability of h-Mb. The SPD concentration effect on the thermodynamic equilibria (K_U) of h-Mb was translated to the changes in preferential interaction coefficient (ΔΓ₂₃) and hydration number (ΔΓ_w). The finding of the positive value of ΔΓ₂₃ and the negative value of ΔΓ_w suggests that the interaction of SPD with h-Mb and the exclusion of water from the protein reduce the conformational stability of h-Mb. Analysis of SPD effects on enthalpy-entropy plots and temperature dependence of unfolding free energy of h-Mb suggests that the SPD forms soft, attractive enthalpic interactions with h-Mb. Analysis of SPD impact on the urea-concentration-dependent thermal unfolding midpoint temperature showed that SPD exhibits an additive effect to the urea-mediated decrease in the thermal stability of h-Mb.