Membrane protein reconstitution into liposomes guided by dual-color fluorescence cross-correlation spectroscopy

Simeonov, Peter; Werner, Stefan; Haupt, Caroline; Tanabe, Mikio; Bacia, Kirsten

doi:10.1016/j.bpc.2013.08.003

Cited by 21 publications

(29 citation statements)

References 45 publications

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“…Dual-color fluorescence cross-correlation spectroscopy (FCCS) has been reported of great value for optimizing membrane protein reconstitution processes thanks to its ability to distinguish micelles, liposomes and aggregates in heterogeneous mixtures and by the fact that it allows co-localization of proteins and lipids in the diffusing assemblies41. The disadvantage of the method is its reliance on fluorescence, which requires fluorescent labelling of the protein and the lipids, and limits the time resolution to avoid bleaching.…”

Section: Discussionmentioning

confidence: 99%

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids

Calmet

Maria

Harté

et al. 2016

Sci Rep

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G-protein coupled receptors (GPCRs) are important therapeutic targets since more than 40% of the drugs on the market exert their action through these proteins. To decipher the molecular mechanisms of activation and signaling, GPCRs often need to be isolated and reconstituted from a detergent-solubilized state into a well-defined and controllable lipid model system. Several methods exist to reconstitute membrane proteins in lipid systems but usually the reconstitution success is tested at the end of the experiment and often by an additional and indirect method. Irrespective of the method used, the reconstitution process is often an intractable and time-consuming trial-and-error procedure. Herein, we present a method that allows directly monitoring the reconstitution of GPCRs in model planar lipid membranes. Plasmon waveguide resonance (PWR) allows following GPCR lipid reconstitution process without any labeling and with high sensitivity. Additionally, the method is ideal to probe the lipid effect on receptor ligand binding as demonstrated by antagonist binding to the chemokine CCR5 receptor.

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Section: Discussionmentioning

confidence: 99%

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids

Calmet

Maria

Harté

et al. 2016

Sci Rep

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“…Embedding chemical reactions within liposomes has been a topic of study for several decades and great advances have been made, such as self-assembly of nanoscale protein networks inside liposomes [40] and embedding of functional transmembrane transporter proteins in their membranes [41,42].…”

Section: Embedding Symbols and Reactionsmentioning

confidence: 99%

Towards experimental P-systems using multivesicular liposomes

2019

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P-systems are abstract computational models inspired by the phospholipid bilayer membranes generated by biological cells. Illustrated here is a mechanism by which recursive liposome structures (multivesicular liposomes) may be experimentally produced through electroformation of dipalmitoylphosphatidylcholine (DOPC) films for use in 'real' P-systems. We first present the electroformation protocol and microscopic characterisation of incident liposomes towards estimating the size of computing elements, level of internal compartment recursion, fault tolerance and stability. Following, we demonstrate multiple routes towards embedding symbols, namely modification of swelling solutions, passive diffusion and microinjection. Finally, we discuss how computing devices based on P-systems can be produced and their current limitations. A P-system or 'membrane computer' is an abstract computational model inspired by biological reactions occurring within the confines of phospholipid (PL) membrane-encapsulated living cells and their subcomponents. Briefly, P-systems are envisaged as hypothetical recursive membrane-bound compartments wherein internal symbolic objects, likened to chemicals and catalysts, may react with each other to do computation, the products of which may remain within their compartment or diffuse inwards or outwards through their

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“…a GUVs exhibiting spherical shape, b the AnxA5-membrane interaction with DOPC allowing changes between the internal and external media and subsequent loss of the optical contrast without membrane disruption, c the effect on GUV membrane morphology after TNAP interaction, showing excess area and filament formation. Magnification ×60, scale bars 20 μm the intrinsic tryptophan enable the making of fluorescence images of the protein-membrane interactions (Simeonov et al 2013). Confocal microscopes are very useful due to their higher spatial resolution when compared to conventional wide field fluorescence microscopes, allowing analysis even with proteoliposomes as small as MVs (Mathiasen et al 2014).…”

Section: Optical Microscopy Applications On Biomineralization Studiesmentioning

confidence: 99%

Biophysical aspects of biomineralization

et al. 2017

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During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix (ECM) by promoting the synthesis of hydroxyapatite (HA) seed crystals in the sheltered interior of membranelimited matrix vesicles (MVs). Several lipid and proteins present in the membrane of the MVs mediate the interactions of MVs with the ECM and regulate the initial mineral deposition and posterior propagation. Among the proteins of MV membranes, ion transporters control the availability of phosphate and calcium needed for initial HA deposition. Phosphatases (orphan phosphatase 1, ectonucleotide pyrophosphatase/ phosphodiesterase 1 and tissue-nonspecific alkaline phosphatase) play a crucial role in controlling the inorganic pyrophosphate/inorganic phosphate ratio that allows MVmediated initiation of mineralization. The lipidic microenvironment can help in the nucleation process of first crystals and also plays a crucial physiological role in the function of MVassociated enzymes and transporters (type III sodiumdependent phosphate transporters, annexins and Na + /K + ATPase). The whole process is mediated and regulated by the action of several molecules and steps, which make the process complex and highly regulated. Liposomes and proteoliposomes, as models of biological membranes, facilitate the understanding of lipid-protein interactions with emphasis on the properties of physicochemical and biochemical processes. In this review, we discuss the use of proteoliposomes as multiple protein carrier systems intended to mimic the various functions of MVs during the initiation and propagation of mineral growth in the course of biomineralization. We focus on studies applying biophysical tools to characterize the biomimetic models in order to gain an understanding of the importance of lipid-protein and lipid-lipid interfaces throughout the process.Keywords Lipid microenvironment . Biomineralization . Matrix vesicles . Liposome . Proteoliposome . Hydroxyapatite Mineralization process and the biogenesis of matrix vesiclesMineralization of cartilage and bone occurs by a series of physicochemical and biochemical processes that together facilitate the deposition of calcium phosphate. The deposited calcium phosphate is subsequently converted into hydroxyapatite (HA) [Ca 10 (PO 4 ) 6 (OH) 2 ] in specific areas of the extracellular matrix (ECM). Various experiments have revealed the presence of HA crystals both inside and outside collagen fibrils in the ECM (McNally et al. 2012) and also within the

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Membrane protein reconstitution into liposomes guided by dual-color fluorescence cross-correlation spectroscopy

Cited by 21 publications

References 45 publications

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids

Towards experimental P-systems using multivesicular liposomes

Biophysical aspects of biomineralization

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