| 1. |
- Bolla, Jani Reddy, et al.
(författare)
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A Mass-Spectrometry-Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins
- 2020
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 59:9, s. 3523-3528
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non-specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid-binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent-resistant lipids bound at the dimer interface in the leucine transporter show decreased k(off) rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid-II results in the formation of a 1:1 protein-lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution.
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| 2. |
- Costeira-Paulo, Joana, et al.
(författare)
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Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase
- 2018
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Ingår i: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 25:3, s. 309-317
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Tidskriftsartikel (refereegranskat)abstract
- The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
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| 3. |
- Gault, Joseph, et al.
(författare)
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Mass Spectrometry Reveals the Direct Action of a Chemical Chaperone
- 2018
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Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 9:14, s. 4082-4086
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Tidskriftsartikel (refereegranskat)abstract
- Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine-N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine-N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.
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| 4. |
- Landreh, Michael, et al.
(författare)
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Effects of Detergent Micelles on Lipid Binding to Proteins in Electrospray Ionization Mass Spectrometry
- 2017
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:14, s. 7425-7430
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Tidskriftsartikel (refereegranskat)abstract
- A wide variety of biological processes rely upon interactions between proteins and lipids, ranging from molecular transport to the organization of the cell membrane. It was recently established that electrospray ionization mass spectrometry (ESI-MS) is capable of capturing transient interactions between membrane proteins and their lipid environment, and a detailed understanding of the underlying processes is therefore of high importance. Here, we apply ESIMS to investigate the factors that govern complex formation in solution and gas phases by comparing nonselective lipid binding with soluble and membrane proteins. We find that exogenously added lipids did not bind to soluble proteins, suggesting that lipids have a low propensity to form electrospray ionization adducts. The presence of detergents at increasing micelle concentrations, on the other hand, resulted in moderate lipid binding to soluble proteins. A direct ESI-MS comparison of lipid binding to the soluble protein serum albumin and to the integral membrane protein NapA shows that soluble proteins acquire fewer lipid adducts. Our results suggest that protein lipid complexes form via contacts between proteins and mixed lipid/detergent micelles. For soluble proteins, these complexes arise from nonspecific contacts between the protein and detergent/lipid micelles in the electrospray droplet. For membrane proteins, lipids are incorporated into the surrounding micelle in solution, and complex formation occurs independently of the ESI process. We conclude that the lipids in the resulting complexes interact predominantly with sites located in the transmembrane segments, resulting in nativelike complexes that can be interrogated by MS.
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| 5. |
- 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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| 6. |
- Landreh, Michael, et al.
(författare)
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Predicting the Shapes of Protein Complexes through Collision Cross Section Measurements and Database Searches
- 2020
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:18, s. 12297-12303
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Tidskriftsartikel (refereegranskat)abstract
- In structural biology, collision cross sections (CCSs) from ion mobility mass spectrometry (IM-MS) measurements are routinely compared to computationally or experimentally derived protein structures. Here, we investigate whether CCS data can inform about the shape of a protein in the absence of specific reference structures. Analysis of the proteins in the CCS database shows that protein complexes with low apparent densities are structurally more diverse than those with a high apparent density. Although assigning protein shapes purely on CCS data is not possible, we find that we can distinguish oblate- and prolate-shaped protein complexes by using the CCS, molecular weight, and oligomeric states to mine the Protein Data Bank (PDB) for potentially similar protein structures. Furthermore, comparing the CCS of a ferritin cage to the solution structures in the PDB reveals significant deviations caused by structural collapse in the gas phase. We then apply the strategy to an integral membrane protein by comparing the shapes of a prokaryotic and a eukaryotic sodium/proton antiporter homologue. We conclude that mining the PDB with IM-MS data is a time- effective way to derive low-resolution structural models.
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| 7. |
- 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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