Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers

Abstract

A new approach for determining the membrane immersion depth of a spin-labeled probe has been developed using paramagnetic relaxation enhancement (PRE) in solid-state NMR spectroscopy. A DOXYL spin label was placed at different sites of 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC) phospholipid bilayers as paramagnetic moieties and the resulting enhancements of the longitudinal relaxation (T₁) times of ³¹P nuclei on the surface of the bilayers were measured by a standard inversion recovery pulse sequence. The ³¹P NMR spin-lattice relaxation times decrease steadily as the DOXYL spin label moves closer to the surface as well as the concentration of the spin-labeled lipids increase. The enhanced relaxation vs. the position and concentration of spin-labels indicate that PRE induced by the DOXYL spin label are significant to determine longer distances over the whole range of the membrane depths. When these data were combined with estimated correlation times τ(c), the r⁻⁶-weighted, time-averaged distances between the spin-labels and the ³¹P nuclei on the membrane surface were estimated. The application of using this solid-state NMR PRE approach coupled with site-directed spin labeling (SDSL) may be a powerful method for measuring membrane protein immersion depth.

Fig. 1

(A) Chemical structures of n-(4,4-dimethyl-oxazolidine-N-oxyl) -palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (n-DOXYL-PSPC, where n=5, 10, 14) and (B) the phospholipid bilayer model. The membrane immersion depth is defined as the shortest distance between the ³¹P and the spin-label DOXYL.

Fig. 2

Spin label - ³¹P distance vs. the correlation time τ_c. Calculated distance changes only in the range of 8 to 13 Å whereas correlation time lies in the range from 0.1 to 25 ns.

Fig. 3

³¹P T₁ vs. the position of DOXYL on PSPC at 5% (molar ratio) of spin-labels. The signal intensity profiles of ³¹P are plotted as a function of the time delay in inversion recovery pulse sequence under control and in the presence of n-DOXYL PSPC. The insert is the typical ³¹P MAS NMR spectrum used to measure ³¹P T₁ by using inversion recovery, with sample spinning speed of 4 KHz at the magic angle.

Fig. 4

Plot of ³¹P T₁ times vs. the concentration of 5-DOXYL-PSPC. A liner relationship (for details see text).

Fig. 5

The concept model of the distance and the immersion depth (not drawn to scale). At a concentration of 5 mol % spin labeled lipid, there is one spin label to relax nearby 20 ³¹P nuclei. According to the area surface and the concentration, the distance between the observed ³¹P and another nearby spin label would be at least ∼ 37 Å away (see text).