Membrane solubilisation and reconstitution by octylglucoside: Comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry

Krylova, Oxana; Jahnke, Nadin; Keller, Sandro

doi:10.1016/j.bpc.2010.03.013

Cited by 19 publications

(26 citation statements)

References 44 publications

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“…Sonicated unilamellar vesicles composed of E. coli polar lipid extract (Avanti Polar Lipids) at a lipid concentration of 28 mg/mL buffer (150 mM KCl, 25 mM Tris, 25 mM Hepes, pH 7.50) were added to the solubilized protein in small steps until reconstitution was complete (44,45). This process was followed by buffer exchange via ultracentrifugation, resuspension of the vesicles, and a final dialysis step.…”

Section: Methodsmentioning

confidence: 99%

Filter gate closure inhibits ion but not water transport through potassium channels

Hoomann

Jahnke

Hörner

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The selectivity filter of K + channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K + hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K + . Here, we show that K + channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K + channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability p f of (6.9 ± 0.6) × 10 −13 cm 3 ·s −1 for both mutants. "Collapse" of the selectivity filter upon K + removal did not alter p f and was fully reversible, as demonstrated by current measurements through planar bilayers in a K + -containing medium to which K + -free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K + in the aqueous solution, which indicates an effective K + dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K + ions in the selectivity filter act to mutually destabilize binding. membrane channels | protein reconstitution | knock-on mechanism | aquaporin | brain water homeostasis

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Section: Methodsmentioning

confidence: 99%

Filter gate closure inhibits ion but not water transport through potassium channels

Hoomann

Jahnke

Hörner

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…A conventional liposome, the SL and SSLs were subjected to Triton X-100 titration and monitored with ITC on a VP-ITC titration calorimeter (GE Healthcare, Uppsala, Sweden) [39] and by DLS. The titrant concentration at the solubilization boundary (C D ) was used to compare the stability of the vesicles.…”

Section: Evaluation Of Liposome Stability By Itc and Dlsmentioning

confidence: 99%

Polyethylene glycol (PEG)-dendron phospholipids as innovative constructs for the preparation of super stealth liposomes for anticancer therapy

Pasut

Paolino

Celia

et al. 2015

Journal of Controlled Release

131

100

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Pegylation of nanoparticles has been widely implemented in the field of drug delivery to prevent macrophage clearance and increase drug accumulation at a target site. However, the shielding effect of polyethylene glycol (PEG) is usually incomplete and transient, due to loss of nanoparticle integrity upon systemic injection. Here, we have synthesized unique PEG-dendron-phospholipid constructs that form super stealth liposomes (SSLs). A β-glutamic acid dendron anchor was used to attach a PEG chain to several distearoyl phosphoethanolamine lipids, thereby differing from conventional stealth liposomes where a PEG chain is attached to a single phospholipid. This composition was shown to increase liposomal stability, prolong the circulation half-life, improve the biodistribution profile and enhance the anticancer potency of a drug payload (doxorubicin hydrochloride).

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“…39 The first injections gave rise to endothermic, smoothly decaying heat signals, which reflected the dissolution of injected vesicles and the incorporation of lipid into mixed micelles. The SOL boundary was reached at a lipid concentration of 3.1 mM, where the micelles became saturated with lipid.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously performed such reconstitution experiments on E. coli polar lipid extract/OG mixtures over a range of lipid and detergent concentrations. 39 Solubilization experiments corresponding to reverse titrations of micelles into vesicles could confirm that this system is under thermodynamic control, allowing for an equilibrium phase diagram to be constructed (cf. Figure 3 below).…”

Section: Resultsmentioning

confidence: 99%

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Real-Time Monitoring of Membrane-Protein Reconstitution by Isothermal Titration Calorimetry

et al. 2013

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Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K+ channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-β-d-glucopyranoside. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration calorimetry can be exploited to monitor the progress of membrane-protein reconstitution in real time, in a noninvasive manner, and at high resolution to yield functional proteoliposomes with a narrow size distribution for further downstream applications.

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Membrane solubilisation and reconstitution by octylglucoside: Comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry

Cited by 19 publications

References 44 publications

Filter gate closure inhibits ion but not water transport through potassium channels

Filter gate closure inhibits ion but not water transport through potassium channels

Polyethylene glycol (PEG)-dendron phospholipids as innovative constructs for the preparation of super stealth liposomes for anticancer therapy

Real-Time Monitoring of Membrane-Protein Reconstitution by Isothermal Titration Calorimetry

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