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

Invisible liposomes: Refractive index matching with sucrose enables flow dichroism assessment of peptide orientation in lipid vesicle membrane
approved September 26, 2002 (received for review April 26, 2002)
Published online before print November 6, 2002
Malin Ardhammar , Per Lincoln, and Bengt Nord?n
Department of Physical Chemistry, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
Communicated by Josef Michl, University of Colorado, Boulder, CO
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* and * transitions of the amide chromophore of the polypeptide backbone could be studied. The observation of a strongly negative LD for the first exciton transition (204 nm) is consistent with a membrane-spanning orientation of two intertwined parallel gramicidin helices, as predicted by coupled-oscillator theory.
Abbreviations: LD, linear dichroism; HD, helical dimer; DH, double helix
Structural properties of biologically active molecules in membranes are difficult to assess under solution conditions. Most of the traditional experimental methods, such as ESR, NMR, and diffraction techniques, require specific labeling, contrast, crystallization, or very high concentrations, conditions that can be difficult to obtain or that may seriously alter the natural environment of the molecules in question. We recently proposed a method based on polarized-light absorption (linear dichroism, LD) for studying the orientational behavior of molecules in a liposomal membrane. The only prerequisite is that the molecule absorbs in the visible-to-UV region of the spectrum (1). The membrane environment used is that of a lipid vesicle, with the possibility to vary lipid content, buffer conditions, and other membrane components. The liposomes are shear-deformed into ellipsoids in a laminar shear flow, and the molecular orientation can be assessed from LD, the differential absorption between light polarized parallel and perpendicular to the flow direction (LD = Apar ? Aperp). LD reports on the orientation of the electronic transition moments: for example, a transition oriented parallel to the lipid chains will give a negative LD signal, whereas a transition oriented parallel to the lipid-bilayer surface will give positive LD. To further explore the method, a series of ruthenium complexes has been used (2), their advantage being on one hand, a stereochemically well-defined scaffold with tunable membrane interaction properties, and on the other, being chromophores that display well-characterized transitions spanning all three dimensions (Fig. 1).
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.
So far, however, one shortcoming of the method has been the substantial light scattering that the liposomes give rise to, the scattering being strongly polarized and therefore disturbing the LD spectrum. This light scattering, mainly arising from the difference in refractive index between the lipid bilayer of the liposomes and the surrounding buffer solution, is difficult to model quantitatively, its origin being the differential scattering of partially aligned ellipsoidal shells in directions parallel and perpendicular to the flow. By shortening the optical path length, the scattering can be reduced so as to allow observation of membrane protein chromophores in the near-UV region (3), but its presence still makes the spectral interpretation difficult.
The principle of using refractive index matching to reduce scattering of biological objects is not new (4). It is widely used to suppress unwanted neutron scattering in small-angle neutron scattering techniques for studying macromolecular systems. More specifically, sucrose has been used as contrast-enhancer in small angle x-ray scattering (5). Sucrose is used for cryoprotection of cell and liposomal solutions and cell-modeling purposes (different forms of saccharides being naturally present at or in the cell membranes), and it is known from calorimetry and x-ray diffraction that sucrose does not significantly alter lipid bilayer packing or phase transition temperature, except at extreme dehydration (6).
Gramicidin has, since the discovery of this antibiotic oligopeptide in 1939 (7), evolved to become an archetype model of a membrane channel. It has a characteristic sequence of alternating D- and L-amino acids and can form two main types of membrane channel or pore structures. Both types consist of two 15-residue peptides, either forming a N-terminal-to-N-terminal helical dimer (HD) of -helices, with a lower helical rise than the common -helix, or forming an intertwined double helix (DH) with parallel peptide -helix chains. Both structures provide a transmembrane pore with a diameter large enough to transport ions (8). The two types can interchange in a bilayer environment, and the equilibrium seems to sensitively depend on bilayer thickness, lipid content, and sample preparation (9).
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