Membrane Orientation Studies with Polarized Light Spectroscopy

Lipid vesicles are versatile tools in modern biophysical science. They can be used as models of the cell membrane, to examine permeability, stability and behavior of molecules in the bilayer. They may act as transporters in a pharmacological context, designed to deliver their cargo to a certain place in the patient. The thesis has a methodological approach to the problem of assessing the orientation of guest molecules in a bilayer environment, a problem pertinent to drug development as well as to the understanding of biochemical and biological processes connected to cell membranes. Lipid vesicles can be deformed in a shear flow into ellipsoidal shapes and these macroscopically oriented samples studied with polarized light. The differential absorption between light polarized parallel and perpendicular to the flow orientation direction tells whether a transition moment in the molecule of interest is oriented preferentially parallel or perpendicular to the lipid chains of the vesicle bilayer. Molecules studied range from small aromatic molecules (pyrene, anthracene) over membrane-spanning substituted aromatic groups to large polypyridyl ruthenium complexes, for which small changes of peripheral substituents are found to cause dramatic changes in the orientational behavior. Refractive index matching of the liposomes, by adding sucrose in the surrounding solution, reduces light scattering dramatically, and for the first time enables study of transitions in the ultraviolet region of the spectrum. An alternative way to achieve a membrane host with anisotropic features is to orient a lamellar lyotropic liquid crystal, consisting of planar bilayers with interstitial water layers, between glass plates. Upon introducing a guest molecule into this system, its orientation can be assessed by tilting the sample and measuring linear dichroism. At normal incidence, the circular dichroism of the oriented molecule can be used to reveal the absolute configuration and as an aid for the assignment of electronic transitions

Membrane Orientation Studies with Polarized Light Spectroscopy

Lipid vesicles are versatile tools in modern biophysical science. They can be used as models of the cell membrane, to examine permeability, stability and behavior of molecules in the bilayer. They may act as transporters in a pharmacological context, designed to deliver their cargo to a certain place in the patient. <p />The thesis has a methodological approach to the problem of assessing the orientation of guest molecules in a bilayer environment, a problem pertinent to drug development as well as to the understanding of biochemical and biological processes connected to cell membranes. Lipid vesicles can be deformed in a shear flow into ellipsoidal shapes and these macroscopically oriented samples studied with polarized light. The differential absorption between light polarized parallel and perpendicular to the flow orientation direction tells whether a transition moment in the molecule of interest is oriented preferentially parallel or perpendicular to the lipid chains of the vesicle bilayer. Molecules studied range from small aromatic molecules (pyrene, anthracene) over membrane-spanning substituted aromatic groups to large polypyridyl ruthenium complexes, for which small changes of peripheral substituents are found to cause dramatic changes in the orientational behavior. Refractive index matching of the liposomes, by adding sucrose in the surrounding solution, reduces light scattering dramatically, and for the first time enables study of transitions in the ultraviolet region of the spectrum. <p />An alternative way to achieve a membrane host with anisotropic features is to orient a lamellar lyotropic liquid crystal, consisting of planar bilayers with interstitial water layers, between glass plates. Upon introducing a guest molecule into this system, its orientation can be assessed by tilting the sample and measuring linear dichroism. At normal incidence, the circular dichroism of the oriented molecule can be used to reveal the absolute configuration and as an aid for the assignment of electronic transitions

Invisible Liposomes: Refractive Index Matching with Sucrose Enables Flow Dichroism Assessment of Peptide Orientation in Lipid Vesicle Membranes

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→π* and π→π* 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