Synergistic interactions of lipids and myelin basic protein

Abstract

This report describes force measurements and atomic force microscope imaging of lipid-protein interactions that determine the structure of a model membrane system that closely mimics the myelin sheath. Our results suggest that noncovalent, mainly electrostatic and hydrophobic, interactions are responsible for the multilamellar structure and stability of myelin. We find that myelin basic protein acts as a lipid coupler between two apposed bilayers and as a lipid "hole-filler," effectively preventing defect holes from developing. From our protein-mediated-adhesion and force-distance measurements, we develop a simple quantitative model that gives a reasonably accurate picture of the molecular mechanism and adhesion of bilayer-bridging proteins by means of noncovalent interactions. The results and model indicate that optimum myelin adhesion and stability depend on the difference between, rather than the product of, the opposite charges on the lipid bilayers and myelin basic protein, as well as on the repulsive forces associated with membrane fluidity, and that small changes in any of these parameters away from the synergistically optimum values can lead to large changes in the adhesion or even its total elimination. Our results also show that the often-asked question of which membrane species, the lipids or the proteins, are the "important ones" may be misplaced. Both components work synergistically to provide the adhesion and overall structure. A better appreciation of the mechanism of this synergy may allow for a better understanding of stacked and especially myelin membrane structures and may lead to better treatments for demyelinating diseases such as multiple sclerosis.

Fig. 1.

Geometry of supported bilayer surfaces during force measurements and AFM imaging. An SFA 2 was used for the force measurements (10, 36). Force measurements were made between two symmetrical, crossed cylindrical surfaces of radius R, which is equivalent to a sphere of radius R approaching a planar surface. Lipid bilayers were formed on freshly cleaved mica substrates by conventional Langmuir–Blodgett deposition (25). (a) The first layer deposited was “solid”-phase dipalmitoylphosphatidylethanolamine (Avanti Polar Lipids) at a surface pressure of 35 mN/m and a molecular area of ≈0.42 nm². The second monolayer was either the control or diseased lipid mixture, given in Table 1, which was deposited at 37 mN/m. After the deposition of the second layer, the bilayer-covered surfaces were transferred underwater into the SFA. The force F between the bilayer-coated surfaces in pH 7.4 4-morpholinepropanesulfonic acid (Mops) buffer (150 mM sodium nitrate/10 mM Mops sodium salt) was measured as a function of separation D, after which the two surfaces were separated, and 100 μl (low coverage) or 200 μl (high coverage) of 0.5 mg/ml solutions of MBP C1 in Mops buffer was injected. (Sodium chloride was substituted by sodium nitrate in the Mops buffer solution to avoid corrosion of the semireflecting silver layers under the mica substrates, which occurs in high concentrations of chloride ions. Mops sodium salt and sodium nitrate were purchased from Sigma–Aldrich.) After allowing the bilayers to equilibrate with the protein for >60 min, the surfaces were brought together again, and the new forces were measured (b). (c) Proposed conformation of the protein interacting with the anionic lipids based on the measured force–distance curves.

Fig. 2.

Measured force profiles between control (a) and diseased (PC-enriched) (b) lipid bilayers with no MBP in 0.15 M NaNO₃ solutions at t = 25°C (cf. Fig. 1a). The right-hand axes give the corresponding energy per unit area between two flat surfaces as given by the Derjaguin approximation (10), E = F/2πR. ○, □, and ▵, approach; •, ▪, and ▴, separation. Three to four separately measured approach and separation runs are shown in each case. The lines are theoretical expressions based on the DLVO theory (9), where the repulsive electrostatic and attractive van der Waals forces are given by F/R = 64πεε₀κ(kT/e)²tanh²(eψ₀/4kT)e^–κD – A/6D², where κ^–1 = 0.8 nm is the expected Debye length in 0.15 M NaNO₃ solution at 25°C(T = 298 K); ψ₀ =–32 and –30 mV, respectively, as calculated for the surface potentials of healthy (a) and diseased (b) bilayers consisting of 5.7% and 5.3% negatively charged lipid (Table 1); and A = 3 × 10^–21 J is the calculated nonretarded but screened Hamaker constant (10). Inserting these values into the above equation yields the curves in a and b, where we have computed the repulsive electrostatic forces on the assumption that the negative charges are located 0.5 nm farther out from the compressed surfaces, which defines D = 0, because the negative charges are located at the extreme ends of the flexible PS^– and CerS^– head groups.

Fig. 3.

Measured force profiles on separation between control (a and c) and diseased (b) bilayers in 0.15 M NaNO₃ solutions at t = 25°C without MBP in the solution (•) and after one injection of 100 μl of 0.5 mg/ml solution of MBP between the surfaces followed by a second injection of the same solution. The forces measured after the first and second injections are identified as “low MBP coverage” (▴) and “high MBP coverage” (▪), respectively. The interactions are purely repulsive on approach, i.e., there is no initial attraction, as also found in the case of the mixed lipid bilayers (Fig. 2). c and its Inset show the low coverage separation force profiles measured between control bilayers, as in a, but in greater detail and showing results from a number of different force runs. Only when the adhesion is not strong enough to cause the surfaces to jump apart can one measure the whole force–distance curve. The straight line through the data points in Inset of c is based on a simple polymer-bridging theory, Eq. 1, as discussed in the text.

Fig. 4.

Tapping-mode AFM images of the same 40 × 40 μm region of a Langmuir–Blodgett-deposited myelin lipid bilayer (control composition from Table 1) on mica imaged in 0.15 mM NaNO₃ solution at 25°C. (a) After 18 min of scanning. (b) After a further 12 h in stagnant water without scanning, showing the growth of 2.7-nm-deep (i.e., monolayer) holes or defects in an otherwise smooth bilayer. (c and d) Images of the same area 30 and 120 min, respectively, after adding 5 μl of 0.5 mg/ml MBP to the solution. MBP adsorbs to the bilayer surface (seen as the small white dots scattered throughout the film) with a lower concentration inside the hydrophobic monolayer holes and a higher concentration at the defect edges, creating a rim 2–5 nm high with the majority of heights being 2 or 4 nm above the surrounding bilayer surface. The defects also gradually heal; the holes fill in, presumably with lipid, because the inside of the hole has the same average height as the outside. The protein rim remains at the original boundaries of the hole. Bilayer deep holes (not shown) also were healed by added MBP. (e) After deposition, stable holes form in the bilayers as in a, but 12 h after myelin lipids with no MBP were added to the buffer solution the holes do not fill in, although some rearrangement of the bilayers has occurred (f). AFM images were acquired by using a Nanoscope III AFM (Digital Instruments, Santa Barbara, CA) with oxide-sharpened silicon nitrate tips with nominal spring constants of ≈0.12 or 0.32 N/m using tapping mode at frequencies of 9 and 29 kHz. The scanning rates ranged between 1 and 4 Hz. Through continuous adjustment of the scanning parameters, we verified that imaging affected neither surface structure nor MBP adsorption dynamics.

Fig. 5.

Total interaction energy, E, per unit membrane area with increasing MBP concentration n₊ for a fixed concentration of negatively charged lipids n_– of 5%. The following values were used: n = 2 × 10¹⁸ per m² (corresponding to 50 Ų per lipid), n_– = 10¹⁷ per m² (corresponding to a 5% fraction of charged lipids), ε_ionic = 2 kT, ε_es = 1 kT, and ε_rep variable between 0 (no thermal repulsion) and 0.1 kT (significant repulsion). Adhesive regimes are shaded.