The lipid environment of Escherichia coli Aquaporin Z

Schmidt, Victoria; Sidore, Marlon; Bechara, Chérine; Duneau, Jean‐Pierre; Sturgis, James N.

doi:10.1016/j.bbamem.2018.10.017

Cited by 36 publications

(39 citation statements)

References 68 publications

(97 reference statements)

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“…CL binding sites are also predicted for a number of other bacterial proteins. 409−412 Likewise, simulation studies on GPCRs are pointing at novel cholesterol binding sites, for instance, in case of the serotonin(1A) receptor 413 the A(2A) adenosine receptor, 414 and the Smoothened receptor, 380 and on the importance of PIP2 in regulating conformational states. 415 Cholesterol binding sites are also found on the Kir2.2 channel, depending on the open/close state of the channel.…”

Section: Increasing Complexitymentioning

confidence: 99%

Computational Modeling of Realistic Cell Membranes

Marrink¹,

Corradi

Souza³

et al. 2019

Chem. Rev.

565

488

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Cell membranes contain a large variety of lipid types and are crowded with proteins, endowing them with the plasticity needed to fulfill their key roles in cell functioning. The compositional complexity of cellular membranes gives rise to a heterogeneous lateral organization, which is still poorly understood. Computational models, in particular molecular dynamics simulations and related techniques, have provided important insight into the organizational principles of cell membranes over the past decades. Now, we are witnessing a transition from simulations of simpler membrane models to multicomponent systems, culminating in realistic models of an increasing variety of cell types and organelles. Here, we review the state of the art in the field of realistic membrane simulations and discuss the current limitations and challenges ahead.

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Section: Increasing Complexitymentioning

confidence: 99%

Computational Modeling of Realistic Cell Membranes

Marrink¹,

Corradi

Souza³

et al. 2019

Chem. Rev.

565

488

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“…Finally, it is possible that the environment of the E. coli inner membrane differentially drives CLC-ec1 dimerization due to changes in lipid composition, or the presence of other unknown cofactors that stabilize equilibrium dimerization in the helix-ala constructs. For example, while 2:1 POPE/POPG is an appropriate synthetic mimic of the overall E. coli polar lipid composition, we know that the biological membranes contain cardiolipin (Ames 1968), possess a large degree of variability in acyl chain composition (Kito et al 1972;Schmidt et al 2019) as well as other lipoidal molecules (Campagna, Miller, and Forman 2003;Robertson 2018). Other environmental factors, such as leaflet asymmetry, ionic conditions and pH could all contribute to shifting the reaction equilibrium in the cell, compared to what we observe in our 2:1 POPE/POPG membranes.…”

Section: Discussionmentioning

confidence: 99%

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

Mersch

Öztürk²,

Park

et al. 2020

Preprint

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CLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of -10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

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“…Thus, it seems more likely that lipid-interactions will be maintained when using SMA than conventional detergents. Several studies have reported analysis of the lipid content of purified protein SMALPs using techniques such as TLC [ 13 , 32 , 37 , 66 , 71 ], HPLC [ 54 ] and/or mass spectrometry [ 22 , 71 , 72 ]. Lipids co-purified from E.coli with SMA-extracted KcsA were found to be enriched in both cardiolipin (CL), and phosphatidylglycerol (PG) [ 13 ], both of which are negatively charged at physiological pH.…”

Section: Protein–lipid Interactionsmentioning

confidence: 99%

Biological insights from SMA-extracted proteins

Unger

Ronco-Campaña

Kitchen

et al. 2021

Biochemical Society Transactions

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In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein–lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein–protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

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The lipid environment of Escherichia coli Aquaporin Z

Cited by 36 publications

References 68 publications

Computational Modeling of Realistic Cell Membranes

Computational Modeling of Realistic Cell Membranes

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

Biological insights from SMA-extracted proteins

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