Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides

Abstract

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.

FIGURE 1

Heat capacity curves obtained by DSC at a scan rate of 30°C/h. (A) Multilamellar C₁₄-peptide/DPPC liposomes containing 0, 5, 10, 15, and 20 mol % of the C₁₄-peptide. Arrows indicate the position of the pretransition. (B) Magnifications of the heat capacity curves shown in A of multilamellar C₁₄-peptide/DPPC liposomes containing 0, 5, 10, and 15 mol % of the C₁₄-peptide.

FIGURE 2

Heat capacity curves obtained by DSC at a scan rate of 30°C/h. (A) Unilamellar DPPC liposomes without (0 mol %) and with 10 mol % of the C₁₄-peptide added to the preformed liposomes from the water phase. (B) Multilamellar DPPC liposomes without and with 10 mol % of the C₁₄-peptide added from the water phase. The inset shows an enlargement of the low-enthalpy transition around 29°C. (C) Multilamellar DPPC liposomes with 10 mol % of the C₁₄-peptide added from the water phase. This liposome suspension was taken through a series of 10 up- and downscans. The first, second, fourth, and 10th upscans are shown.

FIGURE 3

(A) AFM micrograph of a DPPC bilayer in the gel phase at 30°C on a solid support (mica). Defect lines are visible in the DPPC lipid bilayer. (B) The same DPPC bilayer as in A 100 min after C₁₄-peptide addition. It is seen that the C₁₄-peptide predominantly inserts into the defect zones, thereby creating local membrane regions with a high concentration of C₁₄-peptides. The height profile shows that these peptide-enriched membrane regions are ∼10 Å lower than lipid bilayer regions that are unaffected by the peptide.

FIGURE 4

AFM image of supported double bilayers of DPPC lipids incorporated with 10 mol % of the C₁₄-peptide at 32°C. Ripples are seen in the upper bilayer. The height profile corresponds to the white line in the image and shows two characteristic ripple spacings of ∼30 nm and ∼60 nm. These ripple spacings are similar to pure DPPC lipid bilayers.

FIGURE 5

Addition of C₁₄-peptide to supported double bilayers of DPPC lipids in the ripple phase at 37°C. (A) Before addition of the C₁₄-peptide, ripples are present in the upper bilayer island. (B) 15 min after addition of the C₁₄-peptide. The ripples in the upper bilayer island are unaffected by the peptide addition, whereas the structure of the lower DPPC bilayer is clearly affected by the addition of the C₁₄-peptide. (C) 57 min after peptide addition, the ripples on the island have apparently disappeared. The underlying structure of the lower DPPC bilayer is markedly affected by the presence of the C₁₄-peptide. (D) Ripples still present in an island not previously scanned 110 min after C₁₄-peptide addition.

FIGURE 6

(A) Simulation snapshot of the peptide-DPPC system simulated with an applied surface tension of 61 dyn/cm. Modeling and simulation details are provided in Jensen et al. (33). The choline headgroup and the palmitoyl chains are colored yellow and gray, respectively. Water molecules appear in red and white. The peptide-C₁₄ anchor is colored green. The interfacial tryptophan residue is displayed in purple, and the remainder of the peptide appear in ice blue. (B) Equilibrium areas A′(γ) = Ā(DPPC⁺)/Ā(DPPC⁻) ≡ A⁺(γ)/A⁻(γ) of the DPPC bilayer with the anchored peptide (+) relative to pure DPPC lipid bilayers (−) as a function of applied surface tension. Average areas were for all γ computed in the time interval 10–14 ns. Errors in the equilibrium area were obtained as where (C) Average relative deuterium order parameter 〈|SD|′(γ)〉 = 〈|SD|(γ)〉⁺/〈|SD|(γ)〉⁻ of peptide-containing (+) and peptide-free (−) DPPC bilayers displayed as a function of applied surface tension. Averages and errors were computed as in (A). The phase-structure of the bilayer approaches gel- and fluid-like characteristics with decreasing and increasing γ, respectively, in panels B and C.