Lipid-gramicidin interactions using two-dimensional Fourier-transform electron spin resonance

Abstract

The application of two-dimensional Fourier-transform electron-spin-resonance (2D-FT-ESR) to the study of lipid/gramicidin A (GA) interactions is reported. It is shown that 2D-FT-ESR spectra provide substantially enhanced spectral resolution to changes in the dynamics and ordering of the bulk lipids (as compared with cw-ESR spectra), that result from addition of GA to membrane vesicles of dipalmitoylphosphatidylcholine (DPPC) in excess water containing 16-PC as the lipid spin label. The agreement between the theory of Lee, Budil, and Freed and experimental results is very good in the liquid crystalline phase. Both the rotational and translational diffusion rates of the bulk lipid are substantially decreased by addition of GA, whereas the ordering is only slightly increased, for a 1:5 ratio of GA to lipid. The slowing effect on the diffusive rates of adding GA in the gel phase is less pronounced. It is suggested that the spectral fits in this phase would be improved with a more detailed dynamic model. No significant evidence is found in the 2D-FT-ESR spectra for a second immobilized component upon addition of GA, which is in contrast to cw-ESR. It is shown from simulations of the observed 2D-FT-ESR spectra that the additional component seen in cw-ESR spectra, and usually attributed to "immobilized" lipid, is inconsistent with its being characterized by increased ordering, according to a model proposed by Ge and Freed, but it would be consistent with the more conventional model of a significantly reduced diffusional rate. This is because the 2D-FT-ESR spectra exhibit a selectivity, favoring components with longer homogeneous relaxation times, T2. The homogeneous linewidths of the 2D-FT-ESR autopeaks appear to broaden as a function of mixing time. This apparent broadening is very likely due to the process of cooperative order director fluctuations (ODF) of the lipids in the vesicle. This real-time observation of ODF is distinct from, but appears in reasonable agreement with, NMR results. It is found that addition of GA to give the 1:5 ratio has only a small effect on the ODF, but there is a significant temperature dependence.

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

“…In the presence of IB the S c+ and S cÀ signals are quite different, with the S cÀ spectra being substantially sharper due to the echolike cancellation of the IB, which does not occur for the S c+ spectra. In our past studies [7,9] we have found that the S c+ signal is much more attenuated, due to its more rapid decay during t d , because the IB is not refocused. Thus, we make use of the strong and better resolved S cÀ signal.…”

“…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 .…”

“…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.…”

“…(7) or (8) followed by a double Fourier transform, where the real part is taken at each step as summarized in Eq. (9). 4 The experimental result is shown on the left side, with the best fit to a MOMD model shown on the right side.…”

Two-dimensional ELDOR in the study of model and biological membranes

“…These matters ate discussed below. In recent years it has been shown that 2-D ELDOR experiments on spin-labeled lipids in membranes provide much greater spectral resolution to dynamics and ordering in these complex fluids than continuous-wave (CW) ESR spectra are capable of [7,8]. In a study of several different types of spin-labeled lipids in membrane vesicles, it was shown recently that when they are in the ordinary liquid crystalline or liquid-disordered (Ld) phase their 2-D ELDOR spectra are markedly different from those in the liquid-ordered (Lo) phase, which is obtained by adding substantial amounts of cholesterol to the phospholipid.…”