Experimental platform for the functional investigation of membrane proteins in giant unilamellar vesicles

Dolder, Nicolas; Müller, Philipp; Ballmoos, Christoph von

doi:10.1039/d2sm00551d

Cited by 12 publications

(6 citation statements)

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“…For our second control scheme, we reconstituted α -hemolysin in the GUVs. This strategy improved the GUV formation efficiency, as all salts required for transcription could also be supplied after formation, which is beneficial because GUV formation is known to be sensitive to high concentrations of divalent ions [50]. Next, RNA origami production was effectively initiated by adding rNTPs and tracked inside of individual GUVs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells

Tran,

Chakraborty,

Poppleton

et al. 2024

Preprint

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The central dogma at the core of molecular biology states that information flows from DNA to RNA and then to protein. Our research seeks to introduce a conceptually novel approach towards synthetic life by leveraging RNA origami, as an alternative to proteins, requiring only a single copying step between genetic information and function. Here, we report the genetic encoding and expression of an RNA origami cytoskeleton-mimic within giant unilamellar lipid vesicles (GUVs). We design the first RNA origami tiles which fold co-transcriptionally from a DNA template and self-assemble into higher-order 3D RNA origami nanotubes at constant 37◦C in GUVs, where they reach several micrometers in length. Unlike pre-formed and encapsulated DNA cytoskeletons, these GUVs produce their own molecular hardware in an out-of-equilibrium process fuelled by nucleotide feeding. To establish genotype-phenotype correlations, we investigate how sequence mutations govern the contour and persistence length of the RNA origami nanotubes with experiments and coarse-grained molecular-dynamics simulations, realizing a phenotypic transition to closed rings. Finally, we achieve RNA origami cortex formation and GUV deformation without chemical functionalization by introducing RNA aptamers into the tile design.Altogether, this work pioneers the expression of RNA origami-based hardware in vesicles as a new approach towards active, evolvable and RNA-based synthetic cells.

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Section: Resultsmentioning

confidence: 99%

Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells

Tran,

Chakraborty,

Poppleton

et al. 2024

Preprint

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“…GL1 is responsible for the regulation of membrane dynamics in autophagic organelles such as the autophagosome ( Figure 4 a). Recently, the efficient fabrication of proteo-GUVs using polyvinyl alcohol (PVA) or agarose gel [ 81 ] and an electroformation method used Pt wire instead of ITO [ 93 ].…”

Section: Methodology Of Reconstitution Of Membrane Proteins Into Cell...mentioning

confidence: 99%

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes

Tosaka

Kamiya

2023

IJMS

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Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering. However, observing the precise elemental reactions and structures of membrane proteins is difficult, despite their functioning through interactions with various biomolecules in living cells. To investigate these properties, methodologies have been developed to study the functions of membrane proteins that have been purified from biological cells. In this paper, we introduce various methods for creating liposomes or lipid vesicles, from conventional to recent approaches, as well as techniques for reconstituting membrane proteins into artificial membranes. We also cover the different types of artificial membranes that can be used to observe the functions of reconstituted membrane proteins, including their structure, number of transmembrane domains, and functional type. Finally, we discuss the reconstitution of membrane proteins using a cell-free synthesis system and the reconstitution and function of multiple membrane proteins.

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“…Giant unilamellar vesicles (GUVs) integrated with membrane proteins (proteo-GUVs) have curvatures and sizes similar to those of living cells. Therefore, proteo-GUVs may facilitate the investigation of lipid membrane–protein interactions and membrane protein functions, such as enzyme reactions and molecular transportation, using optical microscopy. − A standardized protocol for forming proteo-GUVs has not yet been established because the structures and folding mechanisms of each membrane protein are different. , Several methods for proteo-GUV formation have been proposed, including dehydration–rehydration, − direct (detergent-mediated) reconstitution, − droplet transfer method, , and membrane fusion. , To prepare proteo-GUVs using the dehydration–rehydration method, a dried film containing lipids and membrane proteins is first formed from the nanosized proteoliposome (proteo-LUV) generated by detergent removal and direct incorporation methods . The dried film is then rehydrated using either the electroformation − or gel-assisted swelling method. , In the electroformation method, proteo-GUVs are formed from a dried film on indium tin oxide or platinum wires by adding a low-salt buffer solution and applying an alternating current field.…”

Section: Introductionmentioning

confidence: 99%

“… 7 , 8 Several methods for proteo-GUV formation have been proposed, including dehydration–rehydration, 9 − 11 direct (detergent-mediated) reconstitution, 12 − 15 droplet transfer method, 16 , 17 and membrane fusion. 18 , 19 To prepare proteo-GUVs using the dehydration–rehydration method, a dried film containing lipids and membrane proteins is first formed from the nanosized proteoliposome (proteo-LUV) generated by detergent removal and direct incorporation methods. 20 The dried film is then rehydrated using either the electroformation 21 − 23 or gel-assisted swelling method.…”

Section: Introductionmentioning

confidence: 99%

Formation of Cell-Sized Liposomes Incorporating a β-Barrel-Structured Porin through Rehydration of a Phospholipid-Membrane Protein Dried Film

Tosaka,

Kamiya

2024

ACS Omega

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Giant unilamellar vesicles (GUVs) integrated with membrane proteins (proteo-GUVs) are attractive tools for visualizing membrane protein functions such as enzyme reactions and molecular transportation. In the dehydration−rehydration method, one of the methods used to form proteo-GUVs, they are formed by using a dried film containing phospholipids and membrane proteins through rehydration with an alternating current electric field and a supporting gel. However, these methods make it difficult to form proteo-GUVs under physiological salt concentration and charged phospholipid conditions or carry the risk of gel contamination of lipid membranes. Therefore, proteo-GUVs formed by these rehydration methods may be harmful to membrane proteins. Here, we propose a method for the formation of proteo-GUVs containing physiological salt concentrations and negatively charged phospholipids that do not require an electric field and a supporting gel. To investigate the molecular transport of modified outer membrane protein G (OmpG), OmpG-giant unilamellar vesicles (GUVs) and OmpG-large unilamellar vesicles (LUVs) were formed. The structure and function of different mutants reconstituted into LUVs were evaluated by using circular dichroism spectroscopy and electrophysiological measurements. In addition, the molecular transport of OmpG in GUVs was evaluated by monitoring the Ca 2+ influx into GUVs and fluorescent molecule leakage from GUVs through OmpG nanopores. We found that the amount of Ca 2+ influx into GUVs through the OmpG nanopores depended on the pore size of OmpG. Our method for forming proteo-GUVs can be applied for the functional evaluation of β-barrel porin and in biological sensors using β-barrel porin.

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Experimental platform for the functional investigation of membrane proteins in giant unilamellar vesicles

Abstract: Giant unilamellar vesicles (GUVs) are micrometer-sized model membrane systems that can be viewed directly under the microscope. They serve as scaffolds for the bottom-up creation of synthetic cells, targeted drug...

Cited by 12 publications

References 100 publications

Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells

Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes

Formation of Cell-Sized Liposomes Incorporating a β-Barrel-Structured Porin through Rehydration of a Phospholipid-Membrane Protein Dried Film

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