Invisible liposomes: refractive index matching with sucrose enables flow dichroism assessment of peptide orientation in lipid vesicle membrane.
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.
Ardhammar
Malin
M
Department of Physical Chemistry, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden. maar@phc.chalmers.se
Lincoln
Per
P
Nordén
Bengt
B
D000595:Amino Acid Sequence; D006096:Gramicidin / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D008567:Membranes, Artificial; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000737:chemistry; D012031:Refractometry; D012212:Rheology; D013395:Sucrose / Q000737:chemistry*; D014466:Ultraviolet Rays; D014783:Viscosity
C470087:(12-cyano-12,13-dihydro-11H-cyclopentadipyrido(b-3,2-h, 2',3'-j)phenazine-12-carboxylic acid methyl ester)bis(1,10-phenanthroline)ruthenium(II); D008081:Liposomes; D008565:Membrane Proteins; D008567:Membranes, Artificial; D009942:Organometallic Compounds; D006096:Gramicidin; D013395:Sucrose