Invisible liposomes: refractive index matching with sucrose enables flow dichroism assessment of peptide orientation in lipid vesicle membrane

Abstract

Valuable information on protein-membrane organization may in principle be obtained from polarized-light absorption (linear dichroism, LD) measurement on shear-aligned lipid vesicle bilayers as model membranes. However, attempts to probe LD in the UV wavelength region (<250 nm) have so far failed because of strong polarized light scattering from the vesicles. Using sucrose to match the refractive index and suppress the light scattering of phosphatidylcholine vesicles, we have been able to detect LD bands also in the peptide-absorbing region (200-230 nm). The potential of refractive index matching in vesicle LD as a general method for studying membrane protein structure was investigated for the membrane pore-forming oligopeptide gramicidin incorporated into the liposome membranes. In the presence of sucrose, the LD signals arising from oriented tryptophan side chains as well as from n-->pi* and pi-->pi* transitions of the amide chromophore of the polypeptide backbone could be studied. The observation of a strongly negative LD for the first exciton transition ( approximately 204 nm) is consistent with a membrane-spanning orientation of two intertwined parallel gramicidin helices, as predicted by coupled-oscillator theory.

Fig 1.

Structure of the membrane probe Ru(phen)₂dppzcpCOOCH₃ with the transition moment directions along which the resolved spectral components are polarized (22). Transition moment A coincides with the long-axis of the dppz moiety, B(sh) is directed along the short-axis of the dppz ligand, B(E) is directed between the centers of the two phenanthrolines, and B(A₂) is at an angle of ≈80° to B(E) and perpendicular to the plane in the picture. All B transitions are polarized perpendicular to transition A.

Fig 2.

Approximate transition moment directions of the tryptophan residues used to calculate the expected LD of a gramicidin DH (β DH). The B_b transition is the strongest and occurs close to 220 nm. The L_b transition occurs at slightly longer wavelengths (≈290 nm) than the L_a transition (≈280 nm) (20, 21).

Fig 3.

LD of soy lipid liposomes (sloping spectra) and the membrane probe Ru(phen)₂dppzcpCOOCH₃ in the bilayer of the same liposomes (structured spectra), in the presence of varying sucrose concentrations (0%, 10%, 30%, and 50% wt/wt) in the aqueous buffer. The arrow shows increasing sucrose concentrations.

Fig 4.

LD of gramicidin D in liposomes in the presence (solid line) and absence (dashed line) of 50% sucrose in the buffer. The sample was diluted four times with sucrose buffer to reach 200 nm (dotted line spectrum, scaled). (Inset) The reduced LD (LD/A) of the sucrose-containing sample.

Fig 5.

Calculated coupled-oscillator LD for the peptide π→π* transition of gramicidin in different possible conformations: α-helix (solid line; α), a parallel intertwined double β-helix (long dash; β DH), and a HD of β-helices (short dash; β HD). (Inset) The calculated absorption for the same structures. The π→π* transition moment was taken to be directed along a line connecting the middle of the carbonyl bond and the nitrogen of the amide group (16). An alternative polarization with the transition moment directed between the carbonyl carbon and the nitrogen (17) gave a similar result. The values for the LD and absorption are relative, with the maximal absorbance set to 1.