Translational diffusion and interaction of a photoreceptor and its cognate transducer observed in giant unilamellar vesicles by using dual-focus FCS.
In order to monitor membrane-protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual-focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two-dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D = 2.2 x 10(-8) cm2 s(-1) for the photoreceptor and with D = 4.1 x 10(-8) cm2 s(-1) for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.
Kriegsmann
Jana
J
Research Centre Jülich, ISB 2: Molecular Biophysics, 52425 Jülich, Germany.
Gregor
Ingo
I
von der Hocht
Iris
I
Klare
Johann
J
Engelhard
Martin
M
Enderlein
Jörg
J
Fitter
Jörg
J
D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D002338:Carotenoids / Q000737:chemistry* / Q000378:metabolism; D004058:Diffusion; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D053835:Unilamellar Liposomes / Q000737:chemistry*
D019843:Archaeal Proteins; C434451:HtrII protein, Natronobacterium pharaonis; D008051:Lipid Bilayers; D008565:Membrane Proteins; D020283:Photoreceptors, Microbial; D053835:Unilamellar Liposomes; C434450:phototaxis receptor sensory rhodopsin II, Natronobacterium pharaonis; D002338:Carotenoids