The Puzzling Problem of Cardiolipin Membrane-Cytochrome c Interactions: A Combined Infrared and Fluorescence Study

Abstract

The interaction of cytochrome c (cyt c) with natural and synthetic membranes is known to be a complex phenomenon, involving both protein and lipid conformational changes. In this paper, we combined infrared and fluorescence spectroscopy to study the structural transformation occurring to the lipid network of cardiolipin-containing large unilamellar vesicles (LUVs). The data, collected at increasing protein/lipid ratio, demonstrate the existence of a multi-phase process, which is characterized by: (i) the interaction of cyt c with the lipid polar heads; (ii) the lipid anchorage of the protein on the membrane surface; and (iii) a long-distance order/disorder transition of the cardiolipin acyl chains. Such effects have been quantitatively interpreted introducing specific order parameters and discussed in the frame of the models on cyt c activity reported in literature.

Keywords: cardiolipin; cytochrome c; membrane disorder; protein-membrane binding.

Figure 1

ATR-IR spectrum of large unilamellar vesicles (LUVs) dispersed in PBS buffer (empty points), deconvolved with model functions (dashed lines) and resultant fit curve (red). Comparison with ATR-IR spectrum of cyt c-LUV system (black curve) is reported for sake of clarity (ρ = 0.17).

Figure 2

S² values for the principal spectral bands: (a) ν_s (CH₂) at 2855 cm⁻¹, (b) ν_s (CH₃) at 2873 cm⁻¹, (c) ν_as (CH₂) at 2927 cm⁻¹, and (d) ν (CH=CH) at 3012 cm⁻¹ peaks, as a function of ρ, normalized to the value obtained from the pure LUV spectrum, with exponential fit of the data.

Figure 3

Frequency shift Δν (in cm⁻¹) as a function of ρ: (a) peak at 2855 cm⁻¹, ascribed to CH₂ symmetric stretching mode, with sketches of trans/gauche CL conformers; (b) peak at 2927 cm⁻¹, assigned to CH₂ antisymmetric stretching mode. Data were fitted by sigmoidal curves (shown as guides to the eye) and reported on the same scale.

Figure 4

(a) ATR-IR spectrum of freshly prepared LUV solution; (b) ATR-IR spectra of LUV before (black) and after (pink) cyt c interaction, compared to the spectrum of the pure cyt c (gray). Spectra were normalized to unit area; (c) C-O-P Δν frequency shift (orange circles) and S² values (dark red triangles) normalized to those of LUV solutions as a function of ρ; (d) ${\mathrm{PO}}_2^{-}$ Δν frequency shift (cyan circles) and normalized S² values (blue triangles) of ${\mathrm{PO}}_2^{-}$ groups. In (c,d) panels, data were fitted with exponential curves, reported as dotted guides to eyes.

Figure 5

(a) Laurdan fluorescence as a function of ρ (black line: ρ = 0; red line: ρ = 0.008); in the inset, the initial (black) and final (red) normalized spectra are reported; (b) normalized total fluorescence change of wild type-cyt c (red) and K72N-mutate cyt c (green) as a function of ρ. Solid lines correspond to the best fits (described in Section 4), which yield the dissociation constant values K_{d WT} = (0.18 ± 0.03)·10⁻⁶ M and K_{d K72N} = (0.24 ± 0.03)·10⁻⁶ M, and the number of lipids interacting with each protein molecule N_{L WT} = (21 ± 7) and N_{L K72N} = (24 ± 8), respectively. In the inset, the size distribution of the LUVs obtained by light scattering is reported, in the absence (black points) and in the presence (red points) of wt-cyt c. A representative error bar has been included in each dataset.

Figure 6

(a) Order parameter obtained from the frequency shift Δν (dark yellow) and from the intensities S² (dark red) for the phosphate modes, compared to the normalized FRET data, as a function of ρ; (b) order parameter obtained from the frequency shift Δν (dark yellow) and from the intensities S² (dark red) for the hydrocarbon modes as a function of ρ. Fits to data are reported as dotted lines as guides to eyes.