pH and Temperature-Induced Molten Globule-Like Denatured States of Equinatoxin II: A Study by UV-Melting, DSC, Far- and Near-UV CD Spectroscopy, and ANS Fluorescence

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Abstract
Thermal denaturation of equinatoxin II (EqTxII) in glycine buffer solutions (pH 1.1, 2.0, 3.0, and 3.5) and in triple distilled water (pH 5.5−6.0) was examined by differential scanning calorimetry, UV and CD spectroscopy and fluorescence emission spectroscopy of the added hydrophobic fluorescent probe ANS. At pH 5.5−6.0 and at temperatures below 60 °C, the protein exists in a native state characterized by a pronounced tertiary structure, a β-rich secondary structure and a low degree of ANS-binding. At higher temperatures, it undergoes a two-state conformational transition, (ΔH°)VH = (ΔH°)DSC, into an unfolded state, which is characterized by a complete collapse of its tertiary structure and an incomplete denaturation of its secondary structure. At acidic pH, the EqTxII temperature-induced conformational transition appears at lower temperatures as non-two-state transition accompanied by the formation of an intermediate state which shows characteristics of molten globules, i.e., absence of defined tertiary structure, increase in α-rich secondary structure, and high affinity for ANS. At pH 2.0, the low-temperature initial state of EqTxII is already partially denatured; the tertiary structure is partially disrupted, and a pronounced inequality (ΔH°)VH > (ΔH°)DSC is observed. At pH value of 1.1 and below 60 °C, EqTxII exists in a stable acid-denatured compact state which shows all the characteristics of a molten globule, which even at 95 °C is not completely denatured. According to numerous studies on the pore forming toxins, such acid-denatured compact states may contribute to the protein's ability to penetrate into biological membranes.