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Occupancy distributions of membrane proteins in heterogeneous liposome populations.

ABSTRACT: Studies of membrane protein structure and function often rely on reconstituting the protein into lipid bilayers through the formation of liposomes. Many measurements conducted in proteoliposomes, e.g. transport rates, single-molecule dynamics, monomer-oligomer equilibrium, require some understanding of the occupancy statistics of the liposome population for correct interpretation of the results. In homogenous liposomes, this is easy to calculate as the act of protein incorporation can be described by the Poisson distribution. However, in reality, liposomes are heterogeneous, which alters the statistics of occupancy in several ways. Here, we determine the liposome occupancy distribution for membrane protein reconstitution while considering liposome size heterogeneity. We calculate the protein occupancy for a homogenous population of liposomes with radius r?=?200?nm, representing an idealization of vesicles extruded through 400?nm pores and compare it to the right-skewed distribution of 400?nm 2:1 POPE:POPG vesicles. As is the case for E. coli polar lipids, this synthetic composition yields a sub-population of small liposomes, 25-30?nm in radius with a long tail of larger vesicles. Previously published microscopy data of the co-localization of the CLC-ec1 Cl^-/H⁺ transporter with liposomes, and vesicle occupancy measurements using functional transport assays, shows agreement with the heterogeneous 2:1 POPE:POPG population. Next, distributions of 100?nm and 30?nm extruded 2:1 POPE:POPG liposomes are measured by cryo-electron microscopy, demonstrating that extrusion through smaller pores does not shift the peak, but reduces polydispersity arising from large liposomes. Single-molecule photobleaching analysis of CLC-ec1-Cy5 shows the 30?nm extruded population increases the 'Poisson-dilution' range, reducing the probability of vesicles with more than one protein at higher protein/lipid densities. These results demonstrate that the occupancy distributions of membrane proteins into vesicles can be accurately predicted in heterogeneous populations with experimental knowledge of the liposome size distribution. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

SUBMITTER: Cliff L

PROVIDER: S-EPMC6899186 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Occupancy distributions of membrane proteins in heterogeneous liposome populations.

Cliff Lucy L Chadda Rahul R Robertson Janice L JL

Biochimica et biophysica acta. Biomembranes 20190805 1

Studies of membrane protein structure and function often rely on reconstituting the protein into lipid bilayers through the formation of liposomes. Many measurements conducted in proteoliposomes, e.g. transport rates, single-molecule dynamics, monomer-oligomer equilibrium, require some understanding of the occupancy statistics of the liposome population for correct interpretation of the results. In homogenous liposomes, this is easy to calculate as the act of protein incorporation can be describ ...[more]

PMID: 31394099

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Project description:BackgroundRecently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system.ResultsAtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE.ConclusionFormation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.

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Occupancy distributions of membrane proteins in heterogeneous liposome populations.

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Occupancy distributions of membrane proteins in heterogeneous liposome populations.

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