| 1. |
- Landreh, Michael, et al.
(författare)
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Controlling release, unfolding and dissociation of membrane protein complexes in the gas phase through collisional cooling
- 2015
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Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345 .- 1364-548X. ; 51:85, s. 15582-15584
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry of intact membrane protein complexes requires removal of the detergent micelle by collisional activation. We demonstrate that the necessary energy can be obtained by adjusting the degree of collisional cooling in the ion source. This enables us to extend the energy regime for dissociation of membrane protein complexes.
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| 2. |
- Landreh, Michael, et al.
(författare)
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Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.
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| 3. |
- Yen, Hsin-Yung, et al.
(författare)
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Electrospray ionization of native membrane proteins proceeds via a charge equilibration step
- 2022
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 12:16, s. 9671-9680
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Tidskriftsartikel (refereegranskat)abstract
- Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion–ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.
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