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Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities.

ABSTRACT: Membrane-bound polyprenol-dependent pathways are important for the assembly of essential glycoconjugates in all domains of life. However, despite their prevalence, the functional significance of the extended linear polyprenyl groups in the interactions of the glycan substrates, the biosynthetic enzymes that act upon them, and the membrane bilayer in which they are embedded remains a mystery. These interactions are investigated simultaneously and uniquely through application of the nanodisc membrane technology. The Campylobacter jejuni N-linked glycosylation pathway has been chosen as a model pathway in which all of the enzymes and substrates are biochemically accessible. We present the functional reconstitution of two enzymes responsible for the early membrane-committed steps in glycan assembly. Protein stoichiometry analysis, fluorescence-based approaches, and biochemical activity assays are used to demonstrate the colocalization of the two enzymes in nanodiscs. Isotopic labeling of the substrates reveals that undecaprenyl-phosphate is coincorporated into discs with the two enzymes, and furthermore, that both enzymes are functionally reconstituted and can sequentially convert the coembedded undecaprenyl-phosphate into undecaprenyl-diphosphate-linked disaccharide. These studies provide a proof-of-concept demonstrating that the nanodisc model membrane system represents a promising experimental platform for analyzing the multifaceted interactions among the enzymes involved in polyprenol-dependent glycan assembly pathways, the membrane-associated substrates, and the lipid bilayer. The stage is now set for exploration of the roles of the conserved polyprenols in promoting protein-protein interactions among pathway enzymes and processing of substrates through sequential steps in membrane-associated glycan assembly.

SUBMITTER: Hartley MD

PROVIDER: S-EPMC3876266 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities.

Hartley Meredith D MD Schneggenburger Philipp E PE Imperiali Barbara B

Proceedings of the National Academy of Sciences of the United States of America 20131203 52

Membrane-bound polyprenol-dependent pathways are important for the assembly of essential glycoconjugates in all domains of life. However, despite their prevalence, the functional significance of the extended linear polyprenyl groups in the interactions of the glycan substrates, the biosynthetic enzymes that act upon them, and the membrane bilayer in which they are embedded remains a mystery. These interactions are investigated simultaneously and uniquely through application of the nanodisc membr ...[more]

PMID: 24302767

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Dataset Information

Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities.

Publications

Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities.

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