A Mass‐Spectrometry‐Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Bolla, Jani Reddy; Corey, Robin A.; Sahin, Cagla; Gault, Joseph; Hummer, Alissa; Hopper, Jonathan T. S.; Lane, David P.; Drew, David; Allison, Timothy M.; Stansfeld, Phillip J.; Robinson, Carol V.; Landreh, Michael

doi:10.1002/ange.201914411

Cited by 9 publications

(6 citation statements)

References 24 publications

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“…The identification of specific protein-lipid interactions has been tackled for a number of different proteins. In the well-studied model Gram-negative bacteria Escherichia coli , for instance, which has a relatively simple plasma membrane, the anionic phospholipid cardiolipin (“CDL;” also known as “CL”) has been shown to interact specifically with several membrane proteins, including AmtB ( 2 ), SecYEG ( 3 ), formate dehydrogenase-N ( 4 ), and LeuT ( 5 – 7 ). However, there has been little in the way of systematically modeling CDL interactions with a range of different bacterial proteins in a single study.…”

Section: Introductionmentioning

confidence: 99%

Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

et al. 2021

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Integral membrane proteins are localized and/or regulated by lipids present in the surrounding bilayer. While bacteria have relatively simple membranes, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different Escherichia coli inner membrane proteins. Our data reveal an asymmetry between the membrane leaflets, with increased anionic lipid binding to the inner leaflet regions of the proteins, particularly for cardiolipin. From our simulations, we identify >700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high-affinity cardiolipin binding site on bacterial membrane proteins, paving the way for a heuristic approach to defining other protein-lipid interactions.

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Section: Introductionmentioning

confidence: 99%

Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

et al. 2021

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“…Previous studies show that the exchange between bound lipids and detergent molecules can be used to distinguish non-annular lipid interactions. 31–32 Thus, we added detergents to break up the nanodisc and displace non- or weakly-interacting lipids around AmtB. We tested tetraethylene glycol monooctyl ether (C8E4), Triton X-100, and lauryl dimethyl amine oxide (LDAO) because they require low collisional activation to dissociate from membrane proteins.…”

Section: Resultsmentioning

confidence: 99%

Investigating the Lipid Selectivity of Membrane Proteins in Heterogeneous Nanodiscs

Marty

2021

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The structure and function of membrane proteins can be significantly impacted by the surrounding lipid environment, but membrane protein-lipid interactions in lipid bilayers are often difficult to study due to their transient and polydisperse nature. Here, we used two native mass spectrometry (MS) approaches to investigate how the Escherichia coli ammonium transporter (AmtB) selectively remodels its local lipid environment in heterogeneous lipoprotein nanodiscs. First, we used gas-phase ejection to isolate AmtB with bound lipids from heterogeneous nanodiscs with different combinations of lipids. Second, we used solution-phase detergent flash extraction as an orthogonal approach to study AmtB remodeling with native MS. Flash extraction of AmtB showed that Triton X-100 retains lipid selectivity, but C8E4 distorts preferential lipid interactions. Both approaches reveal that AmtB has a few tight binding sites for PC, is selective for binding PG over-all, and is nonselective for PE, providing a detailed picture of how AmtB binds different lipid head groups in the context of mixed lipid bilayers.

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“…We anticipate that as the resolution of SPA cryo-EM increases for membrane proteins (already typically below 3.5 A ˚), more structures will allow lipid densities in membrane regions to be resolved. For membrane proteins, nMS indicates precisely how many lipid molecules and which lipids are directly involved in membrane protein functionality (Bolla et al, 2020;Gault et al, 2020). Combined with cryo-EM, nMS is poised to become an attractive approach for comprehensive characterization of membrane protein-bound lipid environments (Chorev et al, 2018).…”

Section: Integrative Structural Biology: Combining Output Data From Ms and Cryo-em Shotgun Ms-based Proteomics And Cryo-emmentioning

confidence: 99%

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology

et al. 2022

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Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics. Cryo-EM/ET allows for subnanometer visualization of biological specimens in purified and near-native states, while MS provides bioanalytical information for proteins and protein complexes without introducing additional labels. Here we highlight recent achievements in protein structure and interactome determination using cryo-EM/ET that benefit from additional MS analysis. We also give our perspective on how combining cryo-EM/ET and MS will continue bridging gaps between molecular and cellular studies by capturing and describing 3D snapshots of proteomes and interactomes.

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A Mass‐Spectrometry‐Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Cited by 9 publications

References 24 publications

Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

Investigating the Lipid Selectivity of Membrane Proteins in Heterogeneous Nanodiscs

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology

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