Cytochrome c-lipid interactions: new insights from resonance energy transfer

Abstract

Resonance energy transfer (RET) from anthrylvinyl-labeled phosphatidylcholine (AV-PC) or cardiolipin (AV-CL) to cytochrome c (cyt c) heme moiety was employed to assess the molecular-level details of protein interactions with lipid bilayers composed of PC with 2.5 (CL2.5), 5 (CL5), 10 (CL10), or 20 (CL20) mol % CL under conditions of varying ionic strength and lipid/protein molar ratio. Monte Carlo analysis of multiple data sets revealed a subtle interplay between 1), exchange of the neutral and acidic lipid in the protein-lipid interaction zone; 2), CL transition into the extended conformation; and 3), formation of the hexagonal phase. The switch between these states was found to be controlled by CL content and salt concentration. At ionic strengths ≥ 40 mM, lipid bilayers with CL fraction not exceeding 5 mol % exhibited the tendency to transform from lamellar to hexagonal phase upon cyt c adsorption, whereas at higher contents of CL, transition into the extended conformation seems to become thermodynamically favorable. At lower ionic strengths, deviations from homogeneous lipid distributions were observed only for model membranes containing 2.5 mol % CL, suggesting the existence of a certain surface potential critical for assembly of lipid lateral domains in protein-lipid systems that may subsequently undergo morphological transformations depending on ambient conditions. These characteristics of cyt c-CL interaction are of great interest, not only from the viewpoint of regulating cyt c electron transfer and apoptotic propensities, but also to elucidate the general mechanisms by which membrane functional activities can be modulated by protein-lipid interactions.

Figure 1

Comparison of AV-PC and AV-CL energy transfer profiles obtained for PC/CL liposomes containing 2.5 (A), 5 (B), 10 (C), or 20 (D) mol % CL under conditions of low ionic strength (20 mM). Lipid concentration was 20 μM. Solid lines are to guide the reader's eye.

Figure 2

Effect of lipid concentration on RET in CL_AV-CL model membranes: (A) 2.5 mol % CL, (B) 20 mol % CL. Ionic strength was 20 mM. Solid lines are to guide the reader's eye.

Figure 3

Relative quantum yield of AV-PC (A) and AV-CL (B) in PC/CL (10 mol %) liposomes under conditions of varying ionic strength. Lipid concentration is 20 μM. Solid lines are to guide the reader's eye.

Figure 4

Difference between AV-PC and AV-CL relative quantum yields calculated as a function of total protein concentration for PC/CL model membranes containing 2.5 (A) or 20 (B) mol % CL. Lipid concentration is 20 μM. Solid lines are to guide the reader's eye.

Figure 5

MC calculations of AV-CL relative quantum yield for the cases of the basic lipid demixing model (dash-dot-dotted line), the basic demixing model allowing for complete protein binding (Case I, dash-dotted line), cyt c location in the lipid bilayer center (Case II, dotted line), and aggregation of membrane-bound protein (Case III, dashed line). The experimental curve was obtained for PC/CL (2.5 mol % CL) liposomes at ionic strength 40 mM with donor AV-CL.

Figure 6

Membrane domain characteristics recovered from the combined lipid demixing + extended lipid anchorage MC model for CL10 (squares) and CL20 (circles) systems at ionic strength 40 (upper) and 60 mM (lower).