Fluid mechanical matching of H+-ATP synthase subunit c-ring with lipid membranes revealed by 2H solid-state NMR

Abstract

The F(1)F(o)-ATP synthase utilizes the transmembrane H(+) gradient for the synthesis of ATP. F(o) subunit c-ring plays a key role in transporting H(+) through F(o) in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d(54)) at lipid/protein ratios of 50:1 and 20:1 by means of (2)H-solid-state NMR. In the liquid-crystalline state of DMPC, the (2)H-NMR moment values and the order parameter (S(CD)) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the (2)H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F(1)F(o)-ATPse molecular motor.

FIGURE 1

Analytical ultracentrifugation of subunit c in OG solution above the critical micelle concentration at 20°C. Protein concentration in the cell was scanned using ultraviolet light at 280 nm. (a) Distribution of sedimentation coefficient obtained in the sedimentation velocity experiment at 35,000 rpm for 1 h. (b) Sedimentation equilibrium experiment at 12,000 rpm for 24 h.

FIGURE 2

²H-NMR powder pattern spectra of multilamellar liposomes. (a) Experimental ²H-NMR of fully hydrated DMPC-d₅₄ membranes (black line), DMPC-d₅₄/subunit c membranes (50:1 molar ratio) (red line), and DMPC-d₅₄/subunit c (20:1) membranes (blue line) at 30°C, 25°C, 18°C, 15°C, and −5°C. On the left, theoretical spectra for (b) equally probable three-site jumps corresponding to crankshaft motion of polymethylene chains or threefold rotation of methyl groups on static chain (c) rotation of all-trans polymethylene chain and (d) rigid limit (χ_Q = 167 kHz).

FIGURE 3

Plots of (a) the first moment (M₁) and (b) the second moment (M₂) of the ²H-NMR spectra as a function of temperature on decrease. The error is given by the size of the symbol. Spectra were acquired with decreasing temperature. Dotted lines correspond to the moment values calculated for average order parameters of −1/2 (Fig. 2 c) and −1/3 (Fig. 2 b).

FIGURE 4

(a) De-Paked ²H-NMR spectra of DMPC-d₅₄, DMPC-d₅₄/subunit c (50:1), and DMPC-d₅₄/subunit c (20:1) membranes at 30°C. (b) The segmental order parameter of the C–²H-bond in the fatty acid chains as a function of carbon number i at 30°C. The error is given by the size of the symbol.

FIGURE 5

(a) Partially relaxed de-Paked ²H-NMR spectra of DMPC-d₅₄/subunit c (50:1) at 30°C. Values of inversion-recovery delay τ are 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 ms from bottom to top. (b) The profile of ²H-spin-lattice relaxation rates as a function of segmental position i for DMPC-d₅₄, DMPC-d₅₄/subunit c (50:1), and DMPC-d₅₄/subunit c (20:1) membranes at 30°C. Their errors were ∼9%, 7%, and 4%, respectively.

FIGURE 6

(a) plots as a function of for DMPC-d₅₄, DMPC-d₅₄/subunit c (50:1), and DMPC-d₅₄/subunit c (20:1) membranes at 30°C. Reference data for DMPC-d₅₄/cholesterol (1:1) (30) and DMPC-d₅₄/C₁₂E₈ (2:1) (39) are also presented. (b) A schematic model of the c-ring in lipid bilayers in the liquid-crystalline state. A solution structure of EF_o subunit c (41) was used. Charged amino acid residues are highlighted in the structure of the subunit c-ring (Glu and Asp in red, and Lys and Arg in blue).