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, EqTxII does not exhibit any significant structural changes. Below pH 2, EqTxII undergoes a native-to-partially unfolded transition with a concomitant loss of its rigid tertiary structure and the formation of a non-native secondary structure containing additional alpha-helix. The acid-induced denatured state of EqTxII exhibits a higher intrinsic viscosity and a lower adiabatic compressibility than the native state. Above 50 degrees C, the acid-induced 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 transition occurs between pH 7 and 10 and results in a partial disruption of tertiary structure, while the secondary structure remains largely preserved. The second transition occurs between pH II and 13 and results in the complete loss of tertiary structure and the formation of a non-native, more alpha-helical secondary structure. The acid- and base-induced partially unfolded states of EqTxII form water-soluble oligomers at low salt, while at high salt (> 350 mM NaCl), the acid-induced denatured state precipitates. The hemolytic activity assay shows that the acid- and base-induced denatured states of EqTxII exhibit significantly reduced activity compared to the native state.
Poklar
N
N
Biotechnical Faculty, University of Ljubljana, Slovenia. natasa.poklar@uni-lj.si
Völker
J
J
Anderluh
G
G
Macek
P
P
Chalikia
T V
TV
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D011487:Protein Conformation; D013696:Temperature; D014465:Ultrasonics; D014783:Viscosity
D003064:Cnidarian Venoms; C012307:equinatoxin