Conformational Stability and Hemolytic Activity of Actinoporin RTX-SII from the Sea Anemone Radianthus macrodactylus

Abstract

The spatial organization of actinoporin RTX-SII from the sea anemone Radianthus macrodactylus on the level of tertiary and secondary structures was studied by UV and CD spectroscopy and intrinsic protein fluorescence. The specific and molar extinction coefficients of RTX-SII were determined. The percentages of canonical secondary structures of actinoporin were calculated. The tertiary structure of the polypeptide is well developed and its secondary structure is highly ordered and contains about 50% antiparallel folded beta-sheets. The irreversible thermal denaturation of RTX-SII was studied by CD spectroscopy; a conformational transition occurs at 53 degrees C. Above this temperature irreversible conformational changes are observed in the secondary and tertiary structures. This is accompanied by redistribution of the content of regular and distorted forms of beta-sheet and also by increase in the content of an unordered form. It is suggested that an intermediate is formed in the process of thermal denaturation. Acid-base titration of RTX-SII results in irreversible conformational changes at pH below 2.0 and above 12.0. As shown by intrinsic protein fluorescence, tyrosine residues of RTX-SII make a fundamental contribution to emission, and the total fluorescence depends more on temperature and ionic strength of the solution than tryptophan fluorescence. The data on conformational stability of actinoporin are correlated with data on its hemolytic activity. Activity of RTX-SII significantly decreases at increased temperature and slightly decreases at low pH. Hemolytic activity drastically increases at high pH. Increase in the actinoporin activity at pH above 10 seems to be caused by ionization of the molecule.

Brief heat treatment causes a structural change and enhances cytotoxicity of theEscherichia coliα-hemolysin

“…(45,46) It is well recognized that heat treatment (>65°C) for 30-60 min eliminates the cytotoxicity of RTX toxins. (47) However, we recently demonstrated that a brief heat treatment of the M. haemolytica LKT resulted in greater cytotoxicity, (48) which was accompanied by an irreversible change in secondary structure as determined by circular dichroism (CD) spectroscopy. Varied biophysical approaches have identified other proteins (e.g.…”

Brief heat treatment increases cytotoxicity of Mannheimia haemolytica leukotoxin in an LFA-1 independent manner

“…It has been shown previously that exposure of LKT to high temperature (90–100 °C) for 30 minutes or more, largely eliminates cytotoxic activity for bovine leukocytes [13]. However, other studies showed that exposure of some bacterial toxins to high temperature has varying bio-physical effects on those toxins [22].…”