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Nanodisc-solubilized membrane protein library reflects the membrane proteome

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Abstract

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

A Nanodisc-solubilized membrane protein library is formed by extracting a population of membrane proteins into detergent and then incorporating these proteins into a heterogeneous Nanodisc library, which models the membrane proteome

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Abbreviations

BCA:

Bicinchoninic acid

LC-MS/MS:

Liquid chromatography–tandem mass spectrometry

MP:

Membrane protein

MS:

Mass spectrometry

MSP:

Membrane scaffold protein

Ni-NTA:

Nickel-nitrilotriacetic acid

OG:

Octyl-glucoside

POPC:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SEC:

Size exclusion chromatography

SMPL:

Soluble membrane protein library

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Acknowledgments

We thank Dr. Peter Yau and the University of Illinois Urbana–Champaign Protein Science Facility for assistance in proteomic identification of bands. We thank Michelle Yoo for her assistance in preparing samples. This work was funded by the National Institutes of Health (R01-GM31756 and R01-GM33775 to S.G.S). M.T.M. was supported by the Robert C. and Carolyn J. Springborn Endowment.

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Authors and Affiliations

  1. Department of Chemistry, University of Illinois Urbana–Champaign, Urbana, IL, 61801, USA

    Michael T. Marty & Stephen G. Sligar

  2. Department of Neurobiology, Northwestern University, Evanston, IL, 60208, USA

    Kyle C. Wilcox & William L. Klein

  3. Department of Biochemistry, University of Illinois Urbana–Champaign, Urbana, IL, 61801, USA

    Stephen G. Sligar

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  1. Michael T. Marty
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  2. Kyle C. Wilcox
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  4. Stephen G. Sligar
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Correspondence to Stephen G. Sligar.

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Marty, M.T., Wilcox, K.C., Klein, W.L. et al. Nanodisc-solubilized membrane protein library reflects the membrane proteome. Anal Bioanal Chem 405, 4009–4016 (2013). https://doi.org/10.1007/s00216-013-6790-8

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  • DOI: https://doi.org/10.1007/s00216-013-6790-8

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