Conformation of the gramicidin A channel in phospholipid vesicles: a fluorine-19 nuclear magnetic resonance study.

The membrane conformation of the peptide ionophore gramicidin A is shown by 19F NMR to be described by the N-terminal to N-terminal beta LD helical dimer model proposed by Urry [Urry, D.W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676]. Fully active analogues of gramicidin with 19F labels at both the N- and C-termini are prepared synthetically. Labeled peptides are incorporated into small unilamellar vesicles of dimyristoylphosphatidylcholine. Measurements of the accessibility of the labels to either aqueous or lipophilic paramagnetic probes show that the N-terminus of gramicidin is located in the membrane interior and the C-terminus is at the membrane surface. Of the specific models proposed for the structure of gramicidin, these data are consistent only with that of Urry. The C-terminal 19F NMR peak in vesicles actually consists of three overlapping peaks. Experiments with the aqueous shift reagent Tm3+ show that C-terminal 19F nuclei in the inner and in the outer leaflets of vesicles resonate at different frequencies. The outer leaflet peak in turn consists of two overlapping peaks, possibly due to a local rearrangement of the C-terminal label.

Lateral mobility of phospholipid and cholesterol in the human erythrocyte membrane: effects of protein-lipid interactions.

The phospholipid and cholesterol derivatives N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and N1-cholesterylcarbamoyl-N8-(7-nitro-2,1,3-benzoxadiazol-4-yl )-3,6-dioxaoctane-1 , 8-diamine (NBD-Chol), respectively, were incorporated into egg phosphatidylcholine/cholesterol multilamellar liposomes, human erythrocyte ghost membranes, and multilamellar liposomes derived from extracted human erythrocyte membrane lipids. The lateral mobility of these probes in the plane of the various membranes was measured by using the fluorescence photo-bleaching recovery technique. NBD-PE and NBD-Chol manifested identical lateral mobilities in egg phosphatidylcholine/cholesterol multilamellar liposomes over the range of temperatures from 10 to 37 degrees C and the range of cholesterol mole fractions from 0.0 to 0.5, and in erythrocyte ghost membranes and erythrocyte membrane lipid-derived multilamellar liposomes over the range of temperatures from 15 to 37 degrees C. The weak temperature dependence of the lateral diffusion coefficients of the lipid probes in both artificial and erythrocyte ghost membranes is consistent with the lack of a phase transition in any of these systems over the temperature range studied. Both NBD-PE and NBD-Chol diffuse 4-fold faster in liposomes derived from extracted erythrocyte membrane lipids (D = 8.0 X 10(-9) cm2 s-1 at 37 degrees C) than in the ghost membranes themselves (D = 2.1 X 10(-9) cm2 s-1 at 37 degrees C), suggesting a significant restriction of lipid lateral mobility by membrane protein in the human erythrocyte membrane.

Use of a fluorescent cholesterol derivative to measure lateral mobility of cholesterol in membranes.

N1-Cholesterylcarbamoyl-N8-(4-nitrobenzo-2-oxa-1,3-diazole)-3,6-dioxaoctyl-1,8-diamine (NBD-Chol), a new fluorescent derivative of cholesterol, was incorporated into L-alpha-dimyristoylphosphatidylcholine (Myr2PtdCho)-based liposomes. The lateral mobility of this derivative, as well as that of N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine (NBD-PtdEtn), was measured by fluorescence recovery after photobleaching techniques. In Myr2PtdCho liposomes, the diffusion coefficients (D) of the two probes are the same within experimental error below (D, approximately equal to 2 X 10(-10) cm2 X sec-1) and above (D, approximately equal to 2 X 10(-8) cm2 X sec-1) the main phase transition temperature of the bulk lipid (Tm). There is, however, a distinct difference between the mobilities of the derivatives at concentrations of added cholesterol between 5 and 20 mol % at temperatures below the main phase transition. Under these conditions, the diffusion coefficient of NBD-Chol is approximately twice that of NBD-PtdCho, a result consistent with the idea that cholesterol undergoes a lateral phase separation in these membranes at concentrations less than 20 mol %. At cholesterol concentrations greater than 20 mol % or temperatures above the Tm, the D values of the two probes are identical. The lateral mobility of a cholesterol derivative has thus been monitored directly in cholesterol-containing membranes.

Preparation and properties of O-dansyltyrosine gramicidin C.

Gramicidins A, B, and C are a family of poly-peptide antibiotics which facilitate the passive diffusion of alkali cations and protons through lipid bilayer membranes. It is clear that gramicidin forms a multimeric transmembrane channel and it has been suggested that the channel is an io-conducting dimer in equilibrium on the membrane with non-conducting monomer. We describe the preparation and purification of a derivative of gramicidin C in which the phenolic hydroxyl of the tyrosine at position 11 has been esterified to 8-dimethylaminonaphthalene-1-sulfonate (dansyl). This derivative fluoresces strongly in the visible with an emission maximun in dioxane of 530 nm, an emission lifetime of 16 ns, and a quantum yield of 0.8. Veatch et al. ((1975),J. Mol. Biol. 99, 75) have shown this 0-dansyltyrosine gamicidin C to be a fully active analogue of gramicidin A in artificial lipid bilayer membranes. We here utilize this derivative to further characterize the state of aggregation and rotational mobility of the four interconvertible conformational species formed by gramicidin in nonpolar organic solvents (Veatch et al. (1974), Biochemsitry 13, 5249; Veatch and Blout (1974), Biochemistry 13, 5257). Fluorescence energy transfer from the tryptophans of gramicidin A to the 0-dansyltyrosine of this derivatives supports the conclusion that all of these gramicidin isolated species are aggregates. Decay of fluorescence polarization anisotropy measurements yield a rotational correlation time of 1 ns for the 0-dansyltyrosine chromophore in ethanol in good agreement with the more detailed information previously obtained by 13C-nuclear magnetic resonance for the monomer in dimethyl sulfoxide (Fossel et al. (1974), Biochemistry 13, 5264). However, it is likely that the chromophore has much more rotational mobility than the rest of the gramicidin molecule in the aggregated comformational states.