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.
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 degrees C, the protein exists in a native state characterized by a pronounced tertiary structure, a beta-rich secondary structure and a low degree of ANS-binding. At higher temperatures, it undergoes a two-state conformational transition, (delta H degree)VH = (delta H degree)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 alpha-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 (delta H degree)VH > (delta H degree)DSC is observed. At pH value of 1.1 and below 60 degrees C, EqTxII exists in a stable acid-denatured compact state which shows all the characteristics of a molten globule, which even at 95 degrees 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.
Poklar
N
N
Department of Chemistry, University of Ljubljana, Slovenia.
Lah
J
J
Salobir
M
M
Macek
P
P
Vesnaver
G
G
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003064:Cnidarian Venoms / Q000737:chemistry*; D003603:Cytotoxins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012615:Sea Anemones; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D003064:Cnidarian Venoms; D003603:Cytotoxins; C012307:equinatoxin