Lipid-conjugated fluorescent pH sensors for monitoring pH changes in reconstituted membrane systems.
Accurate real-time measurements of the dynamics of proton concentration gradients are crucial for detailed molecular studies of proton translocation by membrane-bound enzymes. To reduce complexity, these measurements are often carried out with purified, reconstituted enzyme systems. Yet the most paramount problem to detect pH changes in reconstituted systems is that soluble pH reporters leak out of the vesicle system during the reconstitution procedure. This requires loading of substantial amounts of pH-sensors into the lumen of unilamellar liposomes during reconstitution. Here, we report the synthesis and detailed characterisation of two lipid-linked pH sensors employing amine-reactive forms of seminaphthorhodafluors (SNARF®-1 dye) and rhodamine probes (pHrodo™ Red dye). Lipid-conjugation of both dyes allowed for efficient detergent-based reconstitution of these pH indicators into liposomes. Vesicle-embedded pHrodo™ displayed excellent photostability and an optimal pH-response between 4 and 7. The suitability of the lipid-linked pHrodo™ probe as a pH reporter was demonstrated by assaying the activity of a plant plasma membrane H(+)-ATPase (proton pump) reconstituted in proteoliposomes.
Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. tgp@plen.ku.dk.
Gerdi Christine
GC
Kemmer
Sidsel Ammitzbøll
SA
Bogh
Michael
M
Urban
Michael G
MG
Palmgren
Tom
T
Vosch
Jürgen
J
Schiller
Thomas
T
Günther Pomorski
D000577:Amides / Q000737:chemistry; D001578:Benzopyrans / Q000138:chemical synthesis / Q000737:chemistry; D019856:Ethanolamine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry*; D009284:Naphthols / Q000138:chemical synthesis / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011522:Protons; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D000577:Amides; D001578:Benzopyrans; D005456:Fluorescent Dyes; D008081:Liposomes; D009284:Naphthols; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011522:Protons; D012235:Rhodamines; C091666:seminaphthorhodaminefluoride; D019856:Ethanolamine; D006180:Proton-Translocating ATPases