Lipid-Gramicidin Interactions: Dynamic Structure of the Boundary Lipid by 2D-ELDOR

Abstract

The use of 2D-electron-electron double resonance (2D-ELDOR) for the characterization of the boundary lipid in membrane vesicles of DPPC and gramicidin A' (GA) is reported. We show that 2D-ELDOR, with its enhanced spectral resolution to dynamic structure as compared with continuous-wave electron spin resonance, provides a reliable and useful way of studying lipid-protein interactions. The 2D-ELDOR spectra of the end-chain spin label 16-PC in DPPC/GA vesicles is composed of two components, which are assigned to the bulk lipids (with sharp auto peaks and crosspeaks) and to the boundary lipids (with broad auto peaks). Their distinction is clearest for higher temperatures and higher GA concentrations. The quantitative analysis of these spectra shows relatively faster motions and very low ordering for the end chain of the bulk lipids, whereas the boundary lipids show very high "y-ordering" and slower motions. The y-ordering represents a dynamic bending at the end of the boundary lipid acyl chain, which can then coat the GA molecules. These results are consistent with the previous studies by Ge and Freed (1999) using continuous-wave electron spin resonance, thereby supporting their model for GA aggregation and H(II) phase formation for high GA concentrations. Improved instrumental and simulation methods have been employed.

2D-ELDOR using full Sc− fitting and absorption lineshapes

“…It is important to note that in all three steps (i, ii, and iii) a set of 2D-ELDOR spectra for several mixing times, T m are simultaneously fit [3,[6][7][8][9][10][11]. In the full S cÀ method, this set of spectra vs. T m are fit to the same phase corrections, since all the experimental settings are kept constant, except for T m .…”

“…For example, using spin-labeled lipids, it was shown that 2D-ELDOR displays significantly different spectra for the liquid-ordered (L o ) phase vs. the liquid-disordered (L d ) phase, and thus provides a visual distinction of these two important phases [10], since they may be related to the main components found in biomembranes. In another study [11], involving membranes of peptide dissolved in lipid, it was unequivocally shown that there are two distinctly different spectral components, respectively, representing bulk and boundary lipids which coexist [19,20].…”

“…This includes studies of various systems, such as isotropic fluids [1,2], ordered fluids such as liquid crystals [3], peptides [4], polymers [5], complex fluids [6][7][8], model membranes [9][10][11][12], and more recently, biomembranes [13]. As a result, ESR (e.g.…”

Application of the out-of-phase absorption mode to separating overlapping EPR signals with different T1 values

“…The more motionally restricted component (b) is usually assigned to the boundary lipids contacting the GA aggregates [22][23][24][25], in analogy with similar studies on integral transmembrane proteins [26]. The weakly immobilized component (f) corresponds to the fluid (free) lipids, although the EPR spectra of the latter indicate considerably lower rotational mobility as compared with membranes of fluid lipids alone.…”

“…In previous studies [22][23][24][25], mostly on membranes with rather high gramicidin A contents (GA/lipid P 0.1 mol/mol), it was found that the conventional low-power EPR absorption spectra of phospholipids spin-labelled close to the terminal methyl group of the sn-2 chain contain two components. The more motionally restricted component (b) is usually assigned to the boundary lipids contacting the GA aggregates [22][23][24][25], in analogy with similar studies on integral transmembrane proteins [26].…”