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Electroneutral polymer nanodiscs enable interference-free probing of membrane proteins in a lipid-bilayer environment

ABSTRACT: Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that had previously been restricted to soluble or detergent-solubilized proteins. Nonetheless, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques through unspecific interactions. Here we describe a simple strategy for producing water-soluble, electroneutral polymer nanodiscs. Through attachment of a sulfobetaine group, the commercial polymers DIBMA and SMA can be easily converted into the charge-neutral maleimide derivatives, sulf DIBMA and sulf SMA, which readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutrality of the new nanodiscs averts unspecific interactions, thereby enabling reliable lab-on-a-chip detection of protein/lipid interactions and in vitro translation of membrane proteins. Finally, we create a library containing thousands of human membrane proteins and use proteome profiling by mass spectrometry to show the preservation of protein complexes in electroneutral nanodiscs.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast

SUBMITTER: Markus Räschle

LAB HEAD: Markus Räschle

PROVIDER: PXD030958 | Pride | 2023-01-17

REPOSITORIES: Pride

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			Action	DRS
	20191015_HF1_MaRa_SA_P0051_MD_1_50_01.raw	Raw
	20191015_HF1_MaRa_SA_P0051_MD_1_50_02.raw	Raw
	20191015_HF1_MaRa_SA_P0051_MD_1_50_03.raw	Raw
	20191015_HF1_MaRa_SA_P0051_MD_1_50_04.raw	Raw
	20191015_HF1_MaRa_SA_P0051_MD_1_50_05.raw	Raw

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Project description:The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access quantitative database that provides the most efficient extraction conditions of 2065 unique mammalian MPs. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to capture membrane ‘nano-scoops’ containing a target MP efficiently and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

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Electroneutral polymer nanodiscs enable interference-free probing of membrane proteins in a lipid-bilayer environment

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