National Research Council of Canada. NRC Steacie Institute for Molecular Sciences
National Research Council of Canada. NRC Institute for Biological Sciences
The effect of membrane binding on the structure and stability of ferricytochrome c was studied by Fourier-transform infrared spectoscopy and differential scanning calorimetry. Association of cytochrom c with phospholipid membranes containing phosphatidylglycerol as a model acidic phospholipid results in only slight, if any, perturbation of the protein secondary nature. However, upon membrane binding, there is a considerable increase in the accessibility of protein backbone amide groups to hydrogen-deuterium exchange, which suggests a lipid-mediated loosening and/or destabilization o fthe protein tertiary structure. A lipid-induced conformational perturbation of ferricytochrome c is also indicated by a marked decrease in the thermodynamic stability of the membrane-bound protein. Upon binding to the membranes containing dimyristoylphosphatidyglycerol (DMPG) or dioleoylphosphatidylglycerol (DOPG) as a single lipid component, the denaturation temperature of ferricytochrome c decreases by approximately 30⁰C. This is accompanied by a decrease in the calorimetric enthalpy of denaturation, patricularly for the DMPG-associated protein. With ferricytochrome c bound to membranes containing a mixture of DMPG (DOPG) and zwitterionic phosphatidylcholine, the extent of structural perturbation depends on the surface density of the negatively charged lipid head groups, becoming smaller with decreasing proportions of acidic phospholipid in the membrane. The observed destabilization of protein structure mediated by acidic phospholipids (and possibly formation of folding intermediates at the membrane surface) may represent a general property of a larger class of water-soluble proteins for which membrane binding is governed by electrostatic forces.