Synthesis and Functional Reconstitution of Light‐Harvesting Complex II into Polymeric Membrane Architectures

Zapf, Thomas; Tan, Chin Hon; Reinelt, Tobias; Huber, Christoph; Ding, Shiming; Geifman-Shochat, Susana; Paulsen, Harald; Sinner, Eva‐Kathrin

doi:10.1002/anie.201506304

Cited by 11 publications

(9 citation statements)

References 24 publications

(54 reference statements)

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“…Here, we have shown that the titer of a properly folded α-helical membrane protein can be increased by altering membrane concentration and composition in a cell-free reaction. While previous studies have shown that it is possible to cotranslationally fold membrane proteins into diblock copolymer membranes using cell-free expression techniques (34–36), we show that these synthetic membrane amphiphiles can enhance the cotranslational folding and synthesis of a membrane protein, which to the best of our knowledge, has not been previously shown.…”

Section: Discussioncontrasting

confidence: 68%

“…Cell-free methods enable the spontaneous, cotranslational integration of many membrane proteins into vesicles or amphiphilic scaffolds that are present in the reaction (29, 30). A variety of membrane proteins have been expressed into amphiphilic constructs such as lipid vesicles (31–33), polymer scaffolds, polymersomes (15, 16, 34–36), and nanodiscs (37, 38). While these studies suggest that the presence of membranes is important for preventing protein aggregation, an open question remains as to how membrane properties affect protein folding.…”

mentioning

confidence: 99%

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Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

Jacobs

Boyd

Kamat

2019

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

131

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The expression and integration of membrane proteins into vesicle membranes is a critical step in the design of cell-mimetic biosensors, bioreactors, and artificial cells. While membrane proteins have been integrated into a variety of nonnatural membranes, the effects of the chemical and physical properties of these vesicle membranes on protein behavior remain largely unknown. Nonnatural amphiphiles, such as diblock copolymers, provide an interface that can be synthetically controlled to better investigate this relationship. Here, we focus on the initial step in a membrane protein’s life cycle: expression and folding. We observe improvements in both the folding and overall production of a model mechanosensitive channel protein, the mechanosensitive channel of large conductance, during cell-free reactions when vesicles containing diblock copolymers are present. By systematically tuning the membrane composition of vesicles through incorporation of a poly(ethylene oxide)-b-poly(butadiene) diblock copolymer, we show that membrane protein folding and production can be improved over that observed in traditional lipid vesicles. We then reproduce this effect with an alternate membrane-elasticizing molecule, C12E8. Our results suggest that global membrane physical properties, specifically available membrane surface area and the membrane area expansion modulus, significantly influence the folding and yield of a membrane protein. Furthermore, our results set the stage for explorations into how nonnatural membrane amphiphiles can be used to both study and enhance the production of biological membrane proteins.

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Section: Discussioncontrasting

confidence: 68%

mentioning

confidence: 99%

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

Jacobs

Boyd

Kamat

2019

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

131

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“…Cell-free protein synthesis (CFPS) is a relatively new approach , that enables the integration of membrane proteins into model biological membranes without typical constraints. , An important requirement of CFPS is a suitable membrane environment, which is necessary for the proper folding of protein molecules. , So far, the cotranslational integration of membrane proteins into synthetic scaffolds has been demonstrated extensively using liposomes and polymersomes in solution or tethered to a surface. − To adapt this system to be compatible with SLBs requiring a planar geometry, we set out to explore how a cell-free expression system could be leveraged for the cotranslational insertion of membrane proteins into a supported lipid bilayer while maintaining lipid and protein mobility. Specifically, to address the goals of achieving protein mobility in SLBs, we investigated how the incorporation of diblock copolymers into lipid membranes would affect protein properties.…”

Section: Introductionmentioning

confidence: 99%

Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer

Manzer

Ghosh

Jacobs

et al. 2021

ACS Appl. Bio Mater.

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Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.

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“…The wide applicability of this technique was demonstrated with the expression of 85 different endogenous Escherichia coli membrane proteins using IVTT in liposomes [ 14 ]. Polymer membranes are also compatible with IVTT ( figure 2 ), with the expression of Claudin-2 [ 16 ], G-protein-coupled receptors [ 15 , 17 ] and light-harvesting complex II [ 18 ] in polymersomes. However, as both liposomes and polymersomes are static compartments, they lack waste removal or recycling machinery, setting an upper limit to the amount of protein that can be produced before the concentration of inhibitory by-products reaches toxic levels [ 19 ].…”

Section: Compartmentalizationmentioning

confidence: 99%

The hallmarks of living systems: towards creating artificial cells

Yewdall¹,

Mason²,

Hest³

2018

Interface Focus.

134

122

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Despite the astonishing diversity and complexity of living systems, they all share five common hallmarks: compartmentalization, growth and division, information processing, energy transduction and adaptability. In this review, we give not only examples of how cells satisfy these requirements for life and the ways in which it is possible to emulate these characteristics in engineered platforms, but also the gaps that remain to be bridged. The bottom-up synthesis of life-like systems continues to be driven forward by the advent of new technologies, by the discovery of biological phenomena through their transplantation to experimentally simpler constructs and by providing insights into one of the oldest questions posed by mankind, the origin of life on Earth.

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Synthesis and Functional Reconstitution of Light‐Harvesting Complex II into Polymeric Membrane Architectures

Cited by 11 publications

References 24 publications

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression

Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer

The hallmarks of living systems: towards creating artificial cells

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