Acid- and base-induced conformational transitions of equinatoxin II

We have investigated the acid- and base-induced conformational transitions of equinatoxin II (EqTxII), a pore-forming protein, by a combination of CD- spectroscopy, ultrasonic velocimetry, high precision densimetry, viscometry , gel electrophoresis, and hemolytic activity assays. Between pH 7 and 2, E qTxII does not exhibit any significant structural changes. Below pH 2, EqTx II undergoes a native-to-partially unfolded transition with a concomitant l oss of its rigid tertiary structure and the formation of a non-native secon dary structure containing additional alpha -helix, The acid-induced denatur ed state of EqTxII exhibits a higher intrinsic viscosity and a lower adiaba tic compressibility than the native state. Above 50 degreesC, the acid-indu ced denatured state of EqTxII reversibly denatures to a more unfolded state as judged by the far UV CD spectrum of the protein. At alkaline pH, EqTxII undergoes two base-induced conformational transitions. The first transitio n occurs between pH 7 and 10 and results in a partial disruption of tertiar y structure, while the secondary structure remains largely preserved. The s econd transition occurs between pH 11 and 13 and results in the complete lo ss of tertiary structure and the formation of a non-native, more alpha -hel ical secondary structure. The acid- and base-induced partially unfolded sta tes of EqTxII form water-soluble oligomers at low salt, while at high salt (> 350 mM NaCl), the acid-induced denatured state precipitates. The hemolyt ic activity assay shows that the acid- and base-induced denatured states of EqTxII exhibit significantly reduced activity compared to the native state . (C) 2001 Elsevier Science B.V. All rights reserved.