1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology

Tools and Resources
  • Cited 44
  • Views 3,932
  • Annotations
Cite this article as: eLife 2017;6:e20954 doi: 10.7554/eLife.20954

Abstract

Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane integrated enzymes assembling up to hexameric complexes. We further indicate a lipid dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provides a versatile platform for the analysis of membrane proteins in a native environment.

Article and author information

Author details

  1. Erik Henrich

    Institute of Biophysical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  2. Oliver Peetz

    Institute of Physical and Theoretical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  3. Christopher Hein

    Institute of Biophysical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  4. Aisha Laguerre

    Institute of Biophysical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  5. Beate Hoffmann

    Institute of Biophysical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  6. Jan Hoffmann

    Institute of Physical and Theoretical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  7. Volker Dötsch

    Institute of Biophysical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    Competing interests
    Volker Dötsch, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5720-212X
  8. Frank Bernhard

    Institute of Biophysical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  9. Nina Morgner

    Institute of Physical and Theoretical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany
    For correspondence
    morgner@chemie.uni-frankfurt.de
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1872-490X

Funding

Deutsche Forschungsgemeinschaft (Collaborative Research Center (SFB) 807)

  • Erik Henrich
  • Oliver Peetz
  • Christopher Hein

European Strategy Forum on Research Infrastructures (Instruct)

  • Frank Bernhard

Deutsche Forschungsgemeinschaft (DO545/11)

  • Aisha Laguerre

National Institutes of Health (U54GM087519)

  • Beate Hoffmann

Max Planck Research School for Structure and Function of Biological Membranes

  • Beate Hoffmann

Cluster of Excellence Frankfurt

  • Volker Dötsch
  • Nina Morgner

European Research Council (European Union's Seventh Framework Programme (FP7/2007-2013)/ ERC Grant agreement n{degree sign} 337)

  • Nina Morgner

P4EU

  • Frank Bernhard

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Olga Boudker, Weill Cornell Medical College, United States

Publication history

  1. Received: August 25, 2016
  2. Accepted: January 4, 2017
  3. Accepted Manuscript published: January 9, 2017 (version 1)
  4. Version of Record published: January 26, 2017 (version 2)

Copyright

© 2017, Henrich et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,932
    Page views
  • 965
    Downloads
  • 44
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Zachary F Mandell et al.
    Research Article

    NusA and NusG are transcription factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor in B. subtilis, the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, DnusG, and NusA depletion DnusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1400 identified intrinsic terminators. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Loss of NusA and NusG leads to global misregulation of gene expression and loss of NusG results in flagella and swimming motility defects.

    1. Biochemistry and Chemical Biology
    Vidyasiri Vemulapalli et al.
    Research Article Updated

    SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.