Short-chain lipid-conjugated pH sensors for imaging of transporter activities in reconstituted systems and living cells

Kühnel, Ronja Marie; Grifell-Junyent, Marta; Jørgensen, Ida L.; Kemmer, Gerdi Christine; Schiller, Jürgen; Palmgren, Michael G.; Justesen, Bo Højen; Pomorski, Thomas Günther

doi:10.1039/c8an02161a

Cited by 9 publications

(10 citation statements)

References 27 publications

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“…The adaptability of the liposome setup enables facile variations in lipid composition and the incorporation of the pH-sensitive fluorescent dye pHrodo Red via a lipid anchor. The sensor pHrodo Red was selected because it has been employed to report local pH and charge variations ,, on liposomes. The lipid-conjugated pHrodo Red is weakly fluorescent at pH 7.5; because fatty acids are introduced by hydrolase activity, we hypothesized that this would affect the local pH and charges, resulting in a fluorescent-intensity enhancement of the pHrodo sensor (see Figure ).…”

Section: Resultsmentioning

confidence: 99%

“…Expansion of the current assay could allow the parallelized recordings of both function but also molecular recruitment details for additional hydrolytic enzymes, whose substrate can be implemented within membrane surfaces. Dioleoylphosphatidylethanolamine-conjugated pHrodo Red (DOPE-pHrodo Red) was synthesized as described earlier 41 .…”

Section: Discussionmentioning

confidence: 99%

“…Trizma base (T1503; Sigma-Aldrich, Broendby, Denmark), calcium chloride dihydrate (C3306; Sigma-Aldrich), 1,2-di- O -(9 Z -octadecenyl)- sn -glycero-3-phosphocholine (diether-DOPC; 999989; Avanti Polar Lipids, Inc., Alabaster, AL, U.S.A.), 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC; 850375; Avanti Polar Lipids, Inc.), 1,2-dioleoyl- sn -glycero-3-phospho- l -serine (DOPS; 840035C; Avanti Polar Lipids, Inc.), 1,2-disteroyl- sn -glycero-3-phosphoethanolamine- N -[biotinyl(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG 2000 -biotin; Avanti Polar Lipids, Inc.), triolein (1,2,3-tri( cis -9-octadecenoyl)glycerol; T7140; Sigma-Aldrich), and oleic acid (cis-9-octadecenoic acid; O1008; Sigma-Aldrich) were used. Dioleoylphosphatidylethanolamine-conjugated pHrodo Red (DOPE-pHrodo Red) was synthesized as described earlier …”

Section: Methodsmentioning

confidence: 99%

“…Liposomes act as a 3D scaffold for substrate integration while offering an interface for the insertion of a lipase amphipathic helix and interfacial activation ,− ,, even at a relatively low concentration. Upon enzymatic ester hydrolysis, the produced charged carboxylic acids alter the local pH and charges at the membrane surface and, consequently, the fluorescent properties of the pHrodo, which can be quantified reproducibly using fluorescent techniques, as we recently did. ,, …”

Section: Introductionmentioning

confidence: 99%

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Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

et al. 2020

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Lipases are important hydrolytic enzymes used in a spectrum of technological applications, such as the pharmaceutical and detergent industry. Due to their versatile nature and ability to accept a broad range of substrates they have been extensively used for biotechnological and industrial applications. Current assays to measure lipase activity primarily rely on low sensitivity measurement of pH variations or visible changes on material properties, like hydration, and often require high amount of proteins. Fluorescent readouts on the other hand offer high contrast and even single molecule sensitivity, albeit they are reliant on fluorogenic substrates that structurally resemble the native ones. Here we present a method that combines the highly sensitive readout of fluorescent techniques while reporting enzymatic lipase function on native substrates. The method relies on embedding the environmentally sensitive fluorescent dye pHrodo and native substrates into the bilayer of liposomes. The charged products of the enzymatic hydrolysis alter the local membrane environment and thus the fluorescence intensity of pHrodo. The fluorescence can be accurately quantified and directly assigned to product formation and thus enzymatic activity. We illustrated the capacity of the assay to report function of diverse lipases and phospholipases both in a microplate setup and at the single particle level on individual nanoscale liposomes using Total Internal Reflection Fluorescence (TIRF). The parallelized sensitive readout of microscopy combined with the inherent polydispersity in sizes of liposomes allowed us to screen the effect of membrane curvature on lipase function and identify how mutations in the lid region control the membrane curvature dependent activity. We anticipate this methodology to be applicable for sensitive activity readouts for a spectrum of enzymes where the product of enzymatic reaction is charged..

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

et al. 2020

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“…For instance, a large number of fluorescent probes are sensitive to acidic conditions 9‒19 . Some fluorescent probes are responsive from acidic to neutral conditions 20‒26 . Many pH indicators are suitable for monitoring approximately neutral conditions 27‒40 .…”

Section: Introductionmentioning

confidence: 99%

A novel reaction‐based fluorescent probe sensitive to pH changes in multiple intervals

Жен

Zheng

et al. 2020

Coloration Technology

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A new coumarin derivative containing both imine bond and amide moiety was synthesised. It was used as a reaction‐based pH probe and was sensitive to pH changes in the intervals 1.50–4.10 and 10.20–12.86. In the pH range 1.50–4.10, the ultraviolet‐visible (UV‒vis) absorption band of the probe gradually shifted from 353 nm to 340 nm with decrease in the pH value. The colour changed from pale yellow fluorescence at pH 4.10 to blue fluorescence at pH 1.50. Meanwhile the fluorescence emission band at 501 nm gradually shifted to 560 nm accompanying a gradual enhancing in the emission intensity. This pH response is caused by the hydrolysis of the imine bond in the probe under acidic conditions (pH 1.50–4.10). The colourless probe solution turned yellow at pH range 10.20–12.86. Bathochromic shift of the fluorescence band from 501 nm gradually to 550 nm was observed with the increase of pH value from 10.20 to 12.86 accompanying a gradual enhancement of the fluorescence emission intensity, indicating that the new compound serves as a fluorescent pH probe at pH range 10.20–12.86 through a deprotonation process. Under strongly alkaline conditions with pH larger than 12.86, hydrolysis of the lactone structure in the probe occurred to produce non‐fluorescent species.

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A novel colorimetric and ratiometric pH sensor with a notably large Stokes shift for sensing extremely alkaline solution

et al. 2021

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Short-chain lipid-conjugated pH sensors for imaging of transporter activities in reconstituted systems and living cells

Abstract: Short-chain lipid-linked fluorescent pH sensors allow for selective labelling of living cells and liposomes.

Cited by 9 publications

References 27 publications

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

A novel reaction‐based fluorescent probe sensitive to pH changes in multiple intervals

A novel colorimetric and ratiometric pH sensor with a notably large Stokes shift for sensing extremely alkaline solution

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