Structural and thermodynamic characterization of native-like and helical molten globule states of lysozyme: ANS binding and molecular docking insights.

Partially folded protein intermediates can undergo misfolding and aggregation and are often associated with the early stages of neurodegenerative diseases. Understanding the formation of these intermediate states and behaviour under different conditions is therefore important. In this study, acrylamide and iodide (I-) were used as quenchers to investigate structural changes in lysozyme at extreme pH, while guanidinium thiocyanate (GuSCN), 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), and 2,2,2-trifluoroethanol (TFE) were employed to induce conformational changes. Fluorescence, ANS binding, and Förster resonance energy transfer (FRET) studies revealed the molten globule state only at pH 2 in the presence of 0.8 M HFIP and 3 M TFE, whereas GuSCN did not produce such states. Circular dichroism analysis further showed that 0.8 M HFIP generated a native-like molten globule state with a small increase in α-helicity (35-39%), while 3 M TFE produced a highly helical molten globule-like intermediate with a larger increase in α-helicity (35-60%). Thermal unfolding and DSC measurements indicated reduced unfolding cooperativity and altered thermodynamic stability of these intermediate states. TCSPC, ITC, and molecular docking studies further revealed changes in fluorophore accessibility and increased hydrophobic interactions, supporting the molten globule conformations. Overall, the findings suggest that 0.8 M HFIP and 3 M TFE stabilize structurally distinct molten globule-like intermediates of lysozyme at pH 2 and provide insight into early molecular events associated with protein aggregation.