Dataset Information

Reconstitution of proteins on electroformed giant unilamellar vesicles.

ABSTRACT: In vitro reconstitution of simplified biological systems from molecular parts has proven to be a powerful method for investigating the biochemical and biophysical principles underlying cellular processes. In recent years, there has been a growing interest in reconstitution of protein-membrane interactions to understand the critical role played by membranes in organizing molecular-scale events into micron-scale patterns and protrusions. However, while all reconstitution experiments depend on identifying and isolating an essential set of soluble biomolecules, such as proteins, DNA, and RNA, reconstitution of membrane-based processes involves the additional challenge of forming and working with lipid bilayer membranes with composition, fluidity, and mechanical properties appropriate for the process at hand. Here we discuss a selection of methods for forming synthetic lipid bilayer membranes and present a versatile electroformation protocol that our lab uses for reconstituting proteins on giant unilamellar vesicles. This synthetic membrane-based approach to reconstitution offers the ability to study protein organization and activity at membranes under more cell-like conditions, addressing a central challenge to accomplishing the grand goal of "building the cell."

SUBMITTER: Schmid EM

PROVIDER: S-EPMC5564371 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Reconstitution of proteins on electroformed giant unilamellar vesicles.

Schmid Eva M EM Richmond David L DL Fletcher Daniel A DA

Methods in cell biology 20150408

In vitro reconstitution of simplified biological systems from molecular parts has proven to be a powerful method for investigating the biochemical and biophysical principles underlying cellular processes. In recent years, there has been a growing interest in reconstitution of protein-membrane interactions to understand the critical role played by membranes in organizing molecular-scale events into micron-scale patterns and protrusions. However, while all reconstitution experiments depend on iden ...[more]

PMID: 25997355

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Project description:We developed a new (to our knowledge) protocol to generate giant unilamellar vesicles (GUVs) composed of mixtures of single lipopolysaccharide (LPS) species and Escherichia coli polar lipid extracts. Four different LPSs that differed in the size of the polar headgroup (i.e., LPS smooth > LPS-Ra > LPS-Rc > LPS-Rd) were selected to generate GUVs composed of different LPS/E. coli polar lipid mixtures. Our procedure consists of two main steps: 1), generation and purification of oligolamellar liposomes containing LPSs; and 2), electroformation of GUVs using the LPS-containing oligolamellar vesicles at physiological salt and pH conditions. Analysis of LPS incorporation into the membrane models (both oligolamellar vesicles and GUVs) shows that the final concentration of LPS is lower than that expected from the initial E. coli lipids/LPS mixture. In particular, our protocol allows incorporation of no more than 15 mol % for LPS-smooth and LPS-Ra, and up to 25 mol % for LPS-Rc and LPS-Rd (with respect to total lipids). We used the GUVs to evaluate the impact of different LPS species on the lateral structure of the host membrane (i.e., E. coli polar lipid extract). Rhodamine-DPPE-labeled GUVs show the presence of elongated micrometer-sized lipid domains for GUVs containing either LPS-Rc or LPS-Rd above 10 mol %. Laurdan GP images confirm this finding and show that this particular lateral scenario corresponds to the coexistence of fluid disordered and gel (LPS-enriched)-like micron-sized domains, in similarity to what is observed when LPS is replaced with lipid A. For LPSs containing the more bulky polar headgroup (i.e., LPS-smooth and LPS-Ra), an absence of micrometer-sized domains is observed for all LPS concentrations explored in the GUVs (up to ?15 mol %). However, fluorescence correlation spectroscopy (using fluorescently labeled LPS) and Laurdan GP experiments in these microscopically homogeneous membranes suggests the presence of LPS clusters with dimensions below our microscope's resolution (?380 nm radial). Our results indicate that LPSs can cluster into gel-like domains in these bacterial model membranes, and that the size of these domains depends on the chemical structure and concentration of the LPSs.

Dataset Information

Reconstitution of proteins on electroformed giant unilamellar vesicles.

Publications

Reconstitution of proteins on electroformed giant unilamellar vesicles.

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