Partitioning of cytochrome c in multicomponent lipid membranes

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The interaction of water soluble proteins like cytochrome c (cyt c) with lipid membranes plays an important role in a number of biological processes. Cyt c is a positively charged heme protein located at the inner membrane of mitochondria. The main function of this protein is related to electron transport trough the respiratory chain during adenosine triphosphate (ATP) synthesis. cyt c is also one of the pro-apoptotic proteins, which activates the chain of cysteine proteases upon apoptotic stimuli, causing cell death. Thus, the activity of cyt c and the related interactions with membranes are involved at many levels of cell life. Previous studies have shown that at low ionic strength, cyt c inserts into the membrane, while at high ionic strength only a binding process takes place. It has been also demonstrated that insertion of cyt c into charged lipid membranes is stipulated for low lipid-protein ratios. Here we characterized the binding of this protein to multicomponent lipid membranes and resolved the influence of cyt c on the phase state of membranes as well as role of the bilayer surface charge and lipid composition. As a model system, giant unilamellar vesicles (GUVs) were used. To mimic the membrane composition of the inner mitochondrial membrane we employed lipid mixtures of the charged dioleoylphosphatidylglycerol (DOPG), sphingomyelin (SM) and cholesterol. As a first step, we characterized the phase behavior of this mixture from confocal microscopy observations on fluorescently labeled GUVs. Afterwards, we have investigated the alteration of phase behavior of the lipid ternary mixture induced by 0.6 mM Yeast cyt c. The protein concentration as well as the negatively charged lipid component, DOPG were chosen to mimic biologically relevant systems. After characterizing the membrane phase diagram in the presence of cyt c, we focused on the two fluid phase coexistence region, biologically relevant to rafts, and its surrounding environment. We studied the partitioning of cyt c in vesicles which belong to the two fluid phases: liquid ordered (Lo) raft and liquid disordered (Ld), non-raft phases. Analyzes of intensity profiles from fluorescence from the protein partitioning in the two phases yielded partitioning ratios of cyt c in the different domains. Our results indicate that cyt c prefers the DOPG-rich Ld domains, however there is a weak partitioning of cyt c into the Lo domains indicating compositional complexity of the membrane. The specific affinity of the protein to each of the fluid phases and the thermodynamic characterization of these interactions were quantified with isothermal titration calorimetry. We resolved the preferential binding of cyt c to the ternary lipid membranes close either to the Lo or the Ld phases. In parallel, the influence of cyt c on the membrane surface charge and vesicle size was monitored with dynamic light scattering and electrophoretic mobility measurements.