Variation of the Detergent-Binding Capacity and Phospholipid Content of Membrane Proteins When Purified in Different Detergents

İlgü, Hüseyin; Jeckelmann, Jean-Marc; Gachet, María Salomé; Boggavarapu, Rajendra; Ucurum, Zöhre; Gertsch, Jürg; Fotiadis, Dimitrios

doi:10.1016/j.bpj.2014.02.024

Cited by 62 publications

(57 citation statements)

References 43 publications

(41 reference statements)

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“…The mass difference between the peaks corresponded to binding of up to three lipids per protein (Figure b). Increasing the concentration of NG in a stepwise fashion from 0.2 % to 0.5 % led to a gradual removal of lipid adduct peaks, in line with the delipidating properties of NG (Figure b). Together these results show that it is possible to follow lipid exchange with detergent in a concentration‐dependent manner.…”

Section: Figuresupporting

confidence: 55%

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

et al. 2020

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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 koff 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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Section: Figuresupporting

confidence: 55%

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

et al. 2020

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“…Detergents exhibit individual delipidation properties and protein delipidation can also change with the time that is used to expose membrane protein-lipid complexes to detergents 35,36 . From our experience, detergents that co-purify substantial amounts of lipids are often not suitable for the straightforward MS analysis of membrane protein complexes.…”

Section: Resultsmentioning

confidence: 99%

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

et al. 2020

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Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.

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“…AdiC was cloned, overexpressed and purified as described previously (22). SPA experiments using purified AdiC were performed according to ref.…”

Section: Methodsmentioning

confidence: 99%

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

İlgü

Jeckelmann

Gapsys

et al. 2016

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

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Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acidresistance responses, such as the arginine-dependent acid resistance system. In this response, L-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the L-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of L-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli. membrane protein | scintillation proximity assay | substrate binding | transporter | X-ray structure

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Variation of the Detergent-Binding Capacity and Phospholipid Content of Membrane Proteins When Purified in Different Detergents

Cited by 62 publications

References 43 publications

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

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

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

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