Dataset Information

Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions.

ABSTRACT: The ability to rapidly screen interactions between proteins and membrane-like interfaces would aid in establishing the structure-function of protein-lipid interactions, provide a platform for engineering lipid-interacting protein tools, and potentially inform the signaling mechanisms and dynamics of membrane-associated proteins. Here, we describe the preparation and application of water-in-oil (w/o) emulsions with lipid-stabilized droplet interfaces that emulate the plasma membrane inner leaflet with tunable composition. Fluorescently labeled proteins are easily visualized in these synthetic cell-like droplets on an automated inverted fluorescence microscope, thus allowing for both rapid screening of relative binding and spatiotemporally resolved analyses of for example, protein-interface association and dissociation dynamics and competitive interactions, using commonplace instrumentation. We provide protocols for droplet formation, automated imaging assays and analysis, and the production of the positive control protein BcLOV4, a natural photoreceptor with a directly light-regulated interaction with anionic membrane phospholipids that is useful for optogenetic membrane recruitment.

SUBMITTER: Glantz ST

PROVIDER: S-EPMC7185237 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions.

Glantz Spencer T ST Berlew Erin E EE Chow Brian Y BY

Methods in enzymology 20190323

The ability to rapidly screen interactions between proteins and membrane-like interfaces would aid in establishing the structure-function of protein-lipid interactions, provide a platform for engineering lipid-interacting protein tools, and potentially inform the signaling mechanisms and dynamics of membrane-associated proteins. Here, we describe the preparation and application of water-in-oil (w/o) emulsions with lipid-stabilized droplet interfaces that emulate the plasma membrane inner leaflet ...[more]

PMID: 31155055

Similar Datasets

Project description:Adsorption of synthetic alanine-rich peptides to lipid monolayers was studied by X-ray and neutron reflectivity, grazing incidence X-ray diffraction (GIXD), and circular dichroic spectroscopy. The peptides contained histidine residues to drive adsorption to Langmuir monolayers of lipids with iminodiacetate headgroups loaded with Cu2+. Adsorption was found to be irreversible with respect to bulk peptide concentration. The peptides were partially helical in solution at room temperature, the temperature of the adsorption assays. Comparisons of the rate of binding and the structure of the adsorbed layer were made as a function of the number of histidines (from 0 to 2) and also as a function of the positioning of the histidines along the backbone. For peptides containing two histidines on the same side of the helical backbone, large differences were observed in the structure of the adsorbed layer as a function of the spacing of the histidines. With a spacing of 6 A, there was a substantial increase in helicity upon binding (from 17% to 31%), and the peptides adsorbed to a final density approaching that of a nearly completed monolayer of alpha-helices adsorbed side-on. The thickness of the adsorbed layer (17 +/- 2.5 A) was slightly greater than the diameter of alpha-helices, suggesting that the free, unstructured ends extended into solution. With a spacing of 30 A between histidines, a far weaker increase in helicity upon binding was observed (from 13% to 19%) and a much lower packing density resulted. The thickness of the adsorbed layer (10 +/- 4 A) was smaller, consistent with the ends being bound to the monolayer. Striking differences were observed in the interaction of the two types of peptide with the lipid membrane by GIXD, consistent with binding by two correlated sites only for the case of 6 A spacing. All these results are attributed to differences in spatial correlation between the histidines as a function of separation distance along the backbone for these partially helical peptides. Finally, control over orientation was demonstrated by placing a histidine on an end of the sequence, which resulted in adsorbed peptides oriented perpendicular to the membrane.

Dataset Information

Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions.

Publications

Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets