Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs

Hellwig, Nils; Peetz, Oliver; Ahdash, Zainab; Tascón, Igor; Booth, Paula J.; Mikušević, Vedrana; Diskowski, Marina; Politis, Argyris; Hellmich, Yvonne; Hänelt, Inga; Reading, Eamonn; Morgner, Nina

doi:10.1039/c8cc06284f

Cited by 49 publications

(48 citation statements)

References 20 publications

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“…Our structure shows that KimA forms a homodimer, with each protomer consisting of an Nterminal transmembrane (TM) domain and a C-terminal cytoplasmic domain (Fig. 2a, b), as previously suggested 22 . The dimer is stabilized by the swapping of the cytoplasmic domains, which are placed under the opposite membrane domains via long loops connected to the last TM helix (Fig.…”

Section: Structure Of Dimeric Kima In Inward-occluded Conformationsupporting

confidence: 86%

Structural basis of proton-coupled potassium transport in the KUP family

et al. 2020

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Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the cotransport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K + /H + symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins.

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Section: Structure Of Dimeric Kima In Inward-occluded Conformationsupporting

confidence: 86%

Structural basis of proton-coupled potassium transport in the KUP family

et al. 2020

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“…These results, as well as other controversies in the literature, suggested to us a need to move away from detergent, ideally producing complexes from native membranes without recourse to detergents. While many approaches, when combined with MS are promising in this area (45)—including the styrene-maleic acid lipid particle technology (SMALPs), which requires a low pH step—subsequent release of protein assemblies in the gas phase is not straightforward (46). Approaches that could proceed directly from the membrane to the mass spectrometer, without chemical intervention, would undoubtedly be ideal.…”

Section: Moving Away From Membrane Mimeticsmentioning

confidence: 99%

Mass spectrometry: From plasma proteins to mitochondrial membranes

Robinson

2019

Proc. Natl. Acad. Sci. U.S.A.

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In this Inaugural Article, I trace some key steps that have enabled the development of mass spectrometry for the study of intact protein complexes from a variety of cellular environments. Beginning with the preservation of the first soluble complexes from plasma, I describe our early experiments that capitalize on the heterogeneity of subunit composition during assembly and exchange reactions. During these investigations, we observed many assemblies and intermediates with different subunit stoichiometries, and were keen to ascertain whether or not their overall topology was preserved in the mass spectrometer. Adapting ion mobility and soft-landing methodologies, we showed how ring-shaped complexes could survive the phase transition. The next logical progression from soluble complexes was to membrane protein assemblies but this was not straightforward. We encountered many pitfalls along the way, largely due to the use of detergent micelles to protect and stabilize complexes. Further obstacles presented when we attempted to distinguish lipids that copurify from those that are important for function. Developing new experimental protocols, we have subsequently defined lipids that change protein conformation, mediate oligomeric states, and facilitate downstream coupling of G protein-coupled receptors. Very recently, using a radical method—ejecting protein complexes directly from native membranes into mass spectrometers—we provided insights into associations within membranes and mitochondria. Together, these developments suggest the beginnings of mass spectrometry meeting with cell biology.

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“…Since their introduction, xMALPs have resulted in various exciting findings. For example, they enabled the structure determination of the alternative complex III by cryogenic electron microscopy 5 and the elucidation of naturally occurring oligomeric states of various MPs 6 .…”

Section: Introductionmentioning

confidence: 99%

Lipidomic and in-gel analysis of maleic acid co-polymer nanodiscs reveals differences in composition of solubilized membranes

Barniol‐Xicota

Verhelst

2021

Commun Biol

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Membrane proteins are key in a large number of physiological and pathological processes. Their study often involves a prior detergent solubilization step, which strips away the membrane and can jeopardize membrane protein integrity. A recent alternative to detergents encompasses maleic acid based copolymers (xMAs), which disrupt the lipid bilayer and form lipid protein nanodiscs (xMALPs) soluble in aqueous buffer. Although xMALPs are often referred to as native nanodiscs, little is known about the resemblance of their lipid and protein content to the native bilayer. Here we have analyzed prokaryotic and eukaryotic xMALPs using lipidomics and in-gel analysis. Our results show that the xMALPs content varies with the chemical properties of the used xMA.

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Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs

Abstract: Other than more widely used methods, the use of styrene maleic acid copolymers allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding.

Cited by 49 publications

References 20 publications

Structural basis of proton-coupled potassium transport in the KUP family

Structural basis of proton-coupled potassium transport in the KUP family

Mass spectrometry: From plasma proteins to mitochondrial membranes

Lipidomic and in-gel analysis of maleic acid co-polymer nanodiscs reveals differences in composition of solubilized membranes

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