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 fulllength toxin is consistent with two endothermic transitions. Whereas peak A (T m ‫؍‬ 55°C) most likely corresponds to the receptor/translocation domain, peak B (T m ‫؍‬ 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 (T m ‫؍‬ 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 (Ϸ40%) or its pore-forming fragment (Ϸ80%). 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.

Specific Isotope Labeling of Colicin E1 and B Channel Domains For Membrane Topological Analysis by Oriented Solid‐State NMR Spectroscopy

“…At least one reversible folding intermediate has been discernible also for colicin B by using DSC, fluorescence and CD spectroscopies. [44,45] The data that are presented in Figure 3 agree well with an extended 2D helical array of colicin E1 in the membrane, [46] where the interactions of individual helices are important determinants for their membrane alignment. [47] Notably, the hydrophobic region of colicin E1 is too short to span the POPC bilayer twice in a stable manner, [41,48] and the loop region that connects helices 8 and 9 lacks residues that could stabilize a transmembrane helical loop configuration (Figure 1).…”

“…The proteins adopt a globular structure in solution, which partially or fully unfolds upon membrane insertion. Depending on the environmental conditions and interactions with other proteins or with other colicin domains, [44] the hydrophobic helices 8 and 9 (in red) are oriented parallel or perpendicular to the membrane surface. It remains possible that other helices adopt transmembrane alignments for example, due to transmembrane electric potentials.…”

Thermal stability of human matrix metalloproteinases

“…that obeys pseudo first-order kinetics, and where the temperature dependence of the rate constant k is given by the Arrhenius relation, as described previously [13,15]. These assumptions have been shown to hold for the SERCA and PMCA enzymes in skeletal muscle [13,14], several mammal tissues proteins [15] and for Colicines [16]. The fraction of each component denatured as a function of increasing temperature at a constant rate (f D ) is given as,…”

Advances in membrane receptor screening and analysis

“…Typical applications for DSC include: elucidation of thermally induced structural transitions, estimation of formulation stability, chemical half-life studies, amongst others. Several types of membrane-receptor interactions have been probed using DSC, including the anti-apoptotic protein bcl-2 (del Mar Martinez-Senac et al, 2000), phospholipase A 2 (Hoyrup et al, 2001), the pore-forming proteins equinatoxin II (Poklar et al, 1999) and colicin B (Ortega et al, 2001). (For more detailed reviews see: Clas et al, 1999;Freire, 1995;Jelesarov and Bosshard, 1999;Sanchez-Ruiz, 1995;Sanchez-Ruiz and Mateo, 1987).…”