Interaction of Spin-Labeled Lipid Membranes with Transition Metal Ions

Abstract

The large values of spin relaxation enhancement (RE) for PC spin-labels in the phospholipid membrane induced by paramagnetic metal salts dissolved in the aqueous phase can be explained by Heisenberg spin exchange due to conformational fluctuations of the nitroxide group as a result of membrane fluidity, flexibility of lipid chains, and, possibly, amphiphilic nature of the nitroxide label. Whether the magnetic interaction occurs predominantly via Heisenberg spin exchange (Ni) or by the dipole-dipole (Gd) mechanism, it is essential for the paramagnetic ion to get into close proximity to the nitroxide moiety for efficient RE. For different salts of Ni the RE in phosphatidylcholine membranes follows the anionic Hofmeister series and reflects anion adsorption followed by anion-driven attraction of paramagnetic cations on the choline groups. This adsorption is higher for chaotropic ions, e.g., perchlorate. (A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules.) However, there is no anionic dependence of RE for model membranes made from negatively charged lipids devoid of choline groups. We used Ni-induced RE to study the thermodynamics and electrostatics of ion/membrane interactions. We also studied the effect of membrane composition and the phase state on the RE values. In membranes with cholesterol a significant difference is observed between PC labels with nitroxide tethers long enough vs not long enough to reach deep into the membrane hydrophobic core behind the area of fused cholesterol rings. This study indicates one must be cautious in interpreting data obtained by PC labels in fluid membranes in terms of probing membrane properties at different immersion depths when it can be affected by paramagnetic species at the membrane surface.

Figure 1

Examples of experimental saturation curves in fully hydrated deoxygenated DMPC membranes corresponding to different values of the saturation factor P = γ²T₁T₂^eff. (a) 5-PC, 10 mM nickel perchlorate, P = 0.83 G^–2; (b) 10-PC, 30 mM nickel nitrate, P = 1.57 G^–2; (c) 12-PC, 30 mM nickel chloride, P = 2.58 G^–2; (d) 16-PC, 30 mM nickel sulfate, P = 5.06 G^–2; (e) 16-PC, water with no ions, P = 14.91 G^–2. Red lines are fits of the experimental data to eq 1. T = 39 °C.

Figure 2

Profiles of the saturation factor P = γ²T₁T₂^eff in DMPC membrane for different relaxants vs PC number. P was determined from saturation curves for a series of phospholipids systematically labeled at the sn-2 acyl chain at positions n = 5, 7, 10, 12, 14, and 16. (a) No relaxant, oxygen is removed; (b) 30 mM nickel sulfate added, no oxygen; (c) air oxygen, samples are prepared in aerobic conditions; (d) 30 mM nickel chloride, no oxygen; (e) 10 mM nickel perchlorate, no oxygen. T = 39 °C.

Figure 3

Efficiency of different nickel salts in inducing RE in 5, 7, 10, 12, 14, and 16 PC spin-labels in DMPC membrane at 39 °C, measured as Δ(1/P) = 1/P_ion – 1/P₀, where P_ion and P₀ are the values of the saturation factor P in the presence and absence of relaxant.

Figure 4

(a) ESR spectrum of 5-PC in the presence of Ni(ClO₄)₂ can be derived by convolution of a spectrum in the absence of Ni(ClO₄)₂ with the Lorentzian function. Spectrum in the absence of Ni(ClO₄)₂ (blue); spectrum in the presence of 30 mM Ni(ClO₄)₂ (green); convolution of the spectrum in the absence of Ni(ClO₄)₂ with additional 0.64 G (1/T₂) Lorentzian line width (red). (b) ESR spectrum of 5-PC in the presence of Cu(ClO₄)₂ can be derived by convolution of a spectrum in the presence of Ca(ClO₄)₂ with the Lorentzian function. Spectrum in the presence of 10 mM of Ca(ClO₄)₂ (black); spectrum in the presence of 10 mM Cu(ClO₄)₂ (blue); convolution of the spectrum in the presence of Ca(ClO₄)₂ with additional 0.6 G (1/T₂) Lorentzian line width (red).

Figure 5

Broadening of the central spectral component due to T₂ relaxation enhancement by different nickel salts at 30 mM concentration in the series of PC spin-labels.

Figure 6

(a) Spectra of 5PC in DMPG (0.5 mol %) hydrated with 10 mM solutions of MgSO₄, NiSO₄, or Ni(ClO₄)₂. (b) Spectra of 5PC in DMPG (0.5 mol %) in the presence of 10 mM NiSO₄ or Ni(ClO₄)₂ and 2 M NaCl. (c) Broadening of different n-PC spin-labels in DMPG membranes without NaCl (see (a)) in the presence of 10 mM NiSO₄ or Ni(ClO₄)₂.

Figure 7

Broadening of different n-PC spin-labels in DMPG membranes without NaCl (see Figure 6a) in the presence of 10 mM NiSO₄ or Ni(ClO₄)₂.

Figure 8

(a) Tempone line width in the supernatant obtained from DMPG hydrated by suspending in solutions of Ni(ClO₄)₂ of different concentration as a function of total Ni/DMPG ratio. (b) Broadening of 10PC/DMPG mixture hydrated by different concentrations of Ni(ClO₄)₂ depending on total Ni/DMPG ratio.

Figure 9

Saturation curves for 10PC/DMPC at 39 °C in the presence/absence of 30 mM of NiEDDA complex in the water phase.

Figure 10

Dependence on the concentration of NiCl₂ and Ni(ClO₄)₂ in aqueous phase of the additional relaxation broadening of the EPR spectra of 5-PC in DMPC membranes at 39 °C. The dotted lines show simulations using the model described in the text (Results, subsection 8) with Ni:DMPC stoichiometry 1:2 and following values of binding constants: K_Ni = 0.8 M^–1, K_Cl = 1.7 M^–1, and K_ClO4 = 32 M^–1.

Figure 11

Dependences of the DMPC membrane surface coverage by Ni²⁺ ions on the concentration of NaCl and NaClO₄ in aqueous phase in the presence of 30 mM Ni(ClO₄)₂. T = 39 °C. The surface coverage X_Ni is calculated from line broadenings assuming 2:1 DMPC:Ni stoichiometry. (a) 10PC, addition of NaCl; (b) 5PC, addition of NaClO₄. Experimental data are shown by the small closed circles, and simulations for X_Ni based on the Graham–Poisson–Boltzmann model are shown by solid lines; simulations for Ψ₀ are shown by dotted lines. In these simulations the following values of binding constants are used: K_Ni = 0.8, K_ClO4 = 32, K_Cl = 2, and K_Na = 0.

Figure 12

Broadening of PC labels spectra by Ni(ClO₄)₂ in membranes of DMPC or DMPC with 30 mol % cholesterol: (a) 30 mM and (b) 300 mM Ni(ClO₄)₂.

Figure 13

Relaxation enhancement from 10 mM of Ni(ClO₄)₂ introduced into the water phase of lipid DMPC dispersions as a function of n at the P_β (19 °C) and L_α (39 °C).

Figure 14

Comparative effect of metal ions on relaxation of n-PC spin-labels in the DMPC membrane. The salt concetration is 30 mM.

Figure 15

Effect of 30 and 10 mM of Ni(ClO₄)₂ on the line width of different PC spin-labels (circles) and DPPTC (crosses).

Figure 16

In the case of a random distribution of conformations for spin-labeled tethers, the nitroxide moiety can be found with equal probability in any position inside of the sphere with a radius equal to the tether length in the fully stretched conformation of the acyl chain.

Figure 17

(a) Comparison of Δ(1/P)–n dependences for different ions for n-PC/DMPC at 39 °C. The absolute values of RE are normalized by 1 for 5-PC. One can see less steep slope of the curve for Gd. (b) Broadening of n-PC spin-labels in DMPC by 30 mM Ni(ClO₄)₂ vs 10 mM GdCl₃ and 10 mM MnCl₂. (c) RE determined in saturation experiment for 10 mM GdCl₃ and 10 mM Ni(ClO₄)₂ as Δ(1/P).

Figure 18

Dependences of the normalized integral π∫₀^R(R² – l²) × f(l + a) dl/²/₃πR³ on the spin-labeling position n. R = n × (bond length).