Calorimetric Investigations of the Structural Stability and Interactions of Colicin B Domains in Aqueous Solution and in the Presence of Phospholipid Bilayers

Abstract

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (Tm = 55 °C) most likely corresponds to the receptor/translocation domain, peak B (Tm = 59 °C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 °C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (Tm = 65 °C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B (≈40%) or its pore-forming fragment (≈80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T m = 54 °C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.