Lipid-conjugated fluorescent pH sensors for monitoring pH changes in reconstituted membrane systems

Gerdi Christine Kemmer; Sidsel Ammitzbøll Bogh; Michael Urban; Michael Broberg Palmgren; Tom André Jos Vosch; Jürgen Schiller; Thomas Günther-Pomorski

doi:10.1039/c5an01180a

Lipid-conjugated fluorescent pH sensors for monitoring pH changes in reconstituted membrane systems

Gerdi Christine Kemmer, Sidsel Ammitzbøll Bogh, Michael Urban, Michael Broberg Palmgren, Tom André Jos Vosch, Jürgen Schiller, Thomas Günther-Pomorski

21 Citationer (Scopus)

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Abstract

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.

Originalsprog	Engelsk
Tidsskrift	Analyst
Vol/bind	140
Udgave nummer	18
Sider (fra-til)	6313-6320
Antal sider	8
ISSN	0003-2654
DOI	https://doi.org/10.1039/c5an01180a
Status	Udgivet - 21 sep. 2015

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10.1039/c5an01180a

Lipid-conjugated fluorescent pH sensors for monitoring pH changes in reconstituted membrane systemsForlagets udgivne version, 1,43 MB

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abstract = "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{\textregistered}-1 dye) and rhodamine probes (pHrodo{\texttrademark} Red dye). Lipid-conjugation of both dyes allowed for efficient detergent-based reconstitution of these pH indicators into liposomes. Vesicle-embedded pHrodo{\texttrademark} displayed excellent photostability and an optimal pH-response between 4 and 7. The suitability of the lipid-linked pHrodo{\texttrademark} probe as a pH reporter was demonstrated by assaying the activity of a plant plasma membrane H+-ATPase (proton pump) reconstituted in proteoliposomes.",

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T1 - Lipid-conjugated fluorescent pH sensors for monitoring pH changes in reconstituted membrane systems

AU - Kemmer, Gerdi Christine

AU - Bogh, Sidsel Ammitzbøll

AU - Urban, Michael

AU - Palmgren, Michael Broberg

AU - Vosch, Tom André Jos

AU - Schiller, Jürgen

AU - Günther-Pomorski, Thomas

PY - 2015/9/21

Y1 - 2015/9/21

N2 - 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.

AB - 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.

U2 - 10.1039/c5an01180a

DO - 10.1039/c5an01180a

M3 - Journal article

C2 - 26280031

SN - 0003-2654

VL - 140

SP - 6313

EP - 6320

JO - The Analyst

JF - The Analyst

IS - 18

ER -

Lipid-conjugated fluorescent pH sensors for monitoring pH changes in reconstituted membrane systems

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