Investigating the Molecular Mechanism by which Natural Polyamines Alter the Conformational Stability and Dynamics of Lysozyme

Abstract

This work focuses on how natural polyamines (PAs), such as putrescine (PUT), spermidine (SPD), and spermine (SPM), interact with hen egg white lysozyme (HEWL) and how these interactions alter its conformational stability and dynamics at pH 4.4. Analysis of the effects of PAs on the equilibrium thermodynamic parameters, along with molecular dynamics (MD) simulations of HEWL, revealed that these PAs reduce HEWL's structural stability and increase its conformational fluctuations in the following order: SPM > SPD > PUT. This suggests that the number of amino groups in the PAs plays a critical role in altering the protein's stability and dynamics. Both the experimental and MD simulation studies of HEWL yielded positive preferential interaction coefficients and negative preferential hydration parameters for PAs, following the same order: SPM > SPD > PUT. This finding reveals that the preferential binding of PAs to HEWL, along with alterations in the energetics of the hydration layer, typically alters the protein's conformational stability and dynamics, with a more pronounced effect from PAs containing a greater number of amino groups. Additionally, the analysis of the impact of PAs on enthalpy-entropy plots and unfolding free energy-temperature profiles of HEWL reveals that the binding interactions of PAs to HEWL are predominantly of enthalpic in nature. Furthermore, the examination of PA’s effects on the urea-concentration-dependent melting temperature of HEWL reveals that these PAs have an additive effect on urea's denaturing ability, further reducing the thermal stability of HEWL in the following order: SPM > SPD > PUT. Lastly, the analysis of the effects of PAs on the fluorescence lifetime of HEWL reveals that PAs shorten the average fluorescence of HEWL, suggesting that these PAs form excited-state interactions with HEWL.