Measuring Membrane Protein-Lipid Interactions in Nanodiscs with Native Mass Spectrometry

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

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 m...

Combining Mass Spectrometry and X-Ray Crystallography for Analyzing Native-Like Membrane Protein Lipid Complexes

Membrane proteins represent a challenging family of macromolecules, particularly related to the methodology aimed at characterizing their three-dimensional structure. This is mostly due to their amphipathic nature as well as requirements of ligand bindings to stabilize or control their function. Recently, Mass Spectrometry (MS) has become an important tool to identify the overall stoichiometry ...

Probing protein-protein interactions with mass spectrometry.

Membrane protein assembly into Nanodiscs.

Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane proteins for biophysical, enzymatic or structural investigations. This means for rendering membrane proteins soluble at the single molecule level offers advantages over liposomes or detergent micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structur...

Isolation of yeast complex IV in native lipid nanodiscs.

We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11nm×14nm. Based on this size we estimated that each CytcO was surrounded by ~100 p...