Refractive-index-based screening of membrane-protein-mediated transfer across biological membranes.
Numerous membrane-transport proteins are major drug targets, and therefore a key ingredient in pharmaceutical development is the availability of reliable, efficient tools for membrane transport characterization and inhibition. Here, we present the use of evanescent-wave sensing for screening of membrane-protein-mediated transport across lipid bilayer membranes. This method is based on a direct recording of the temporal variations in the refractive index that occur upon a transfer-dependent change in the solute concentration inside liposomes associated to a surface plasmon resonance (SPR) active sensor surface. The applicability of the method is demonstrated by a functional study of the aquaglyceroporin PfAQP from the malaria parasite Plasmodium falciparum. Assays of the temperature dependence of facilitated diffusion of sugar alcohols on a single set of PfAQP-reconstituted liposomes reveal that the activation energies for facilitated diffusion of xylitol and sorbitol are the same as that previously measured for glycerol transport in the aquaglyceroporin of Escherichia coli (5 kcal/mole). These findings indicate that the aquaglyceroporin selectivity filter does not discriminate sugar alcohols based on their length, and that the extra energy cost of dehydration of larger sugar alcohols, upon entering the pore, is compensated for by additional hydrogen-bond interactions within the aquaglyceroporin pore.
Brändén
Magnus
M
Division of Biological Physics, Chalmers University of Technology, Gothenburg, Sweden. magnus.branden@chalmers.se
Tabaei
Seyed R
SR
Fischer
Gerhard
G
Neutze
Richard
R
Höök
Fredrik
F
D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D004058:Diffusion; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D018272:Porins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D012031:Refractometry; D020349:Surface Plasmon Resonance
D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; C449801:PfAQP protein, Plasmodium falciparum; D018272:Porins; D015800:Protozoan Proteins