Determination of liposome size: A tool for protein reconstitution

Vojta, Aleksandar; Scheuring, Johannes; Neumaier, Nikolaus; Mirus, Oliver; Weinkauf, Sevil; Schleiff, Enrico

doi:10.1016/j.ab.2005.09.003

Cited by 9 publications

(5 citation statements)

References 23 publications

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“…For binding analysis liposomes with a 200 nm diameter were produced as described [ 41 ] in 20 mM Tris/HCl, pH 7,4 and 300 mM sucrose and diluted to a final concentration of 10 mM lipids. For reconstitution 4 mg phosphatidylcholine were resuspended in 400 μl 20 mM Tris/HCl pH 7,4 and 400 mM sucrose and Mega 9 was added to a final concentration of 80 mM.…”

Section: Methodsmentioning

confidence: 99%

The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

et al. 2007

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Background: Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems.

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Section: Methodsmentioning

confidence: 99%

The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

et al. 2007

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“…Proteoliposome Preparation and Flotation Assay-Lipid vesicles (PC, fraction IV, azolectin; Sigma) were prepared as previously reported (18) in buffer containing 10 mM Mops/Tris, 100 mM NaCl, pH 7.0, 1% SDS. Liposome-associated and liposomefree proteins were separated by flotation through a sucrose gradient.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Tic110, a Channel-forming Protein at the Inner Envelope Membrane of Chloroplasts, Unveils a Response to Ca2+ and a Stromal Regulatory Disulfide Bridge

Balsera

Goetze

Kovács-Bogdán

et al. 2009

Journal of Biological Chemistry

117

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Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92-959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca 2؉ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.After the proposed endosymbiotic event that gave rise to chloroplasts and, as a consequence, to the massive transfer of genes from the endosymbiont to the host cell nucleus, two new machineries were developed at the outer and inner envelope of chloroplasts, the TOC and TIC complexes, respectively. The machineries transport plastid proteins, whose synthesis was moved to the cytosol, back to the plastid (for a review, see Refs. 1 and 2). Whereas the channel of the TOC complex is homologous to the Omp85 family of transporters in bacteria (3), the TIC complex shows no homology to known transport systems, since the plasma membrane-bacterial transport machineries were relocated to the thylakoid membrane. Moreover, there was an essential requirement for establishing new lines of communication between the organelle and other parts of the cell (4). In chloroplasts of land plants, redox regulation is linked to oxygen and/or light. The regulation of chloroplast-specific processes by redox in accord with the status of the organelle applies not only for transcription and translation but also for translocation. Redox regulation at the translocation level has been proposed for the TIC machinery on the basis of protein composition and dynamics (5-8) as well as specific in vitro import experiments (9). However, despite advances in our biochemical understanding of the import process, little is presently known regarding details of the role of redox or of the structure of the TIC complex, particularly with respect to its channel subunits.Tic110 is essential for protein import into plastids (10 -12). It has been proposed to be a pore-forming protein and one of the main components of the TIC complex (13). Previous studies have provided information about certain aspects of Tic110 structure-function. The region from residu...

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“…20–25 Droplet interface bilayers have been developed only recently, and are formed spontaneously at the contact point of two water droplets in oil. 26 Liposomes are used widely in drug delivery applications; 27,28 they are also important tools for displaying membrane proteins 29,30 and have been applied in studies of lipid dynamics and phase separation. 31 …”

Section: Introductionmentioning

confidence: 99%

“…They have been used for fundamental studies of lipid bilayer structure , and single-molecule studies of protein electrophysiology. − Supported bilayers involve the fabrication of a lipid bilayer upon a solid surface; they have been broadly applied for the display of membrane proteins and biosensing applications. − Droplet interface bilayers have been developed only recently, and are formed spontaneously at the contact point of two water droplets in oil . Liposomes are used widely in drug delivery applications; , they are also important tools for displaying membrane proteins , and have been applied in studies of lipid dynamics and phase separation …”

Section: Introductionmentioning

confidence: 99%

Microfluidic Fabrication of Asymmetric Giant Lipid Vesicles

Malmstadt

2011

ACS Appl. Mater. Interfaces

123

112

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We have developed a microfluidic technology for the fabrication of compositionally asymmetric giant unilamellar vesicles (GUVs). The vesicles are assembled in two independent steps. In each step, a lipid monolayer is formed at a water-oil interface. The first monolayer is formed inside of a microfluidic device with a multiphase droplet flow configuration consisting of a continuous oil stream in which water droplets are formed. These droplets are dispensed into a vessel containing a layer of oil over a layer of water. The second lipid monolayer is formed by transferring the droplets through this second oil-water interface by centrifugation. By dissolving different lipid compositions in the different oil phases, the composition of each leaflet of the resulting lipid bilayer can be controlled. We have demonstrated membrane asymmetry by showing differential fluorescence quenching of labeled lipids in each leaflet and by demonstrating that asymmetric GUVs will bind an avidin-coated surface only when biotinylated lipids are targeted to the outer leaflet. In addition, we have demonstrated the successful asymmetric targeting of phosphatidylserine lipids to each leaflet, producing membranes with a biomimetic and physiologically relevant compositional asymmetry.

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Determination of liposome size: A tool for protein reconstitution

Cited by 9 publications

References 23 publications

The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

Characterization of Tic110, a Channel-forming Protein at the Inner Envelope Membrane of Chloroplasts, Unveils a Response to Ca2+ and a Stromal Regulatory Disulfide Bridge

Microfluidic Fabrication of Asymmetric Giant Lipid Vesicles

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