Membrane Protein Stabilization Strategies for Structural and Functional Studies

Errasti-Murugarren, Ekaitz; Bartoccioni, Paola; Palacı́n, Manuel

doi:10.3390/membranes11020155

Cited by 26 publications

(35 citation statements)

References 139 publications

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“… 34 Future attempts to achieve this goal will include different lipids and/or variants of MSPs. 35 However, as this approach requires customized solutions rather than being generally applicable to IMPs, we switched to an alternative strategy using the previously described BP-1 peptides to stabilize IMPs in the absence of lipids. To this end, we modified the BP-1 peptide with silica-precipitating peptides based on R5 to obtain functional DGK embedded in silica.…”

Section: Resultsmentioning

confidence: 99%

“…This might be due to the specific properties of DGK and its strong dependence on bilayer thickness and the properties of lipids (lipid mixtures) used in general . Future attempts to achieve this goal will include different lipids and/or variants of MSPs . However, as this approach requires customized solutions rather than being generally applicable to IMPs, we switched to an alternative strategy using the previously described BP-1 peptides to stabilize IMPs in the absence of lipids.…”

Section: Resultsmentioning

confidence: 99%

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Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Bialas

Becker

2021

Bioconjugate Chem.

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Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological application are complicated by the difficulty to stabilize them in solution. We set out to develop a biomimetic procedure to encapsulate functional integral membrane proteins in silica to facilitate their handling under otherwise detrimental conditions and thereby extend their applicability. To this end, we designed and expressed new fusion constructs of the membrane scaffold protein MSP with silica-precipitating peptides based on the R5 sequence from the diatom Cylindrotheca fusiformis . Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that membrane lipid nanodiscs surrounded by our MSP variants fused to an R5 peptide, so-called nanodiscs, were formed. Exposing them to silicic acid led to silica-encapsulated nanodiscs, a new material for stabilizing membrane structures and a first step toward incorporating membrane proteins in such structures. In an alternative approach, four fusion constructs based on the amphiphilic β-sheet peptide BP-1 and the R5 peptide were generated and successfully employed toward silica encapsulation of functional diacylglycerol kinase (DGK). Silica-encapsulated DGK was significantly more stable against protease exposure and incubation with simulated gastric fluid (SGF) and intestinal fluid (SIF).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Bialas

Becker

2021

Bioconjugate Chem.

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“…One AqpZ monomer is originally embedded in a 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid-nanodisc stabilized by the MSP1E2D1 membrane scaffold protein. The diameter of the corresponding nanodisc is 11.1 nm [22]. A cylindrical nanopore is carved out of a silica-cristobalite slab which surface was functionalized with hydroxyl groups (-OH).…”

Section: Resultsmentioning

confidence: 99%

“…One AqpZ monomer was embedded in a 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) lipid-nanodisc stabilized by the MSP1E2D1 membrane scaffold protein. The diameter and net charge of the corresponding nanodisc are 11.1 nm and −14e, respectively [22]. The number of POPC lipid molecules in the discoidal lipid bilayer is 230.…”

Section: System Characteristicsmentioning

confidence: 99%

Biohybrid membrane formation by directed insertion of Aquaporin into a solid-state nanopore

Sicard¹,

Yazaydın²

2022

Preprint

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Technical challenges in molecule sensing and chemical detection have created an increasing demand for transformative materials with high sensitivity and specificity. Biohybrid nanopores have attracted growing interest as they can ideally combine the durability of solid-state nanopores with the precise structure of biological nanopores. Particular care must be taken to control how biological nanopores adapt to their surroundings once in contact with the solid-state nanopore. Two major challenges are to precisely control this adaptability under dynamic conditions and provide predesigned functionalities that can be manipulated for engineering applications. Here, we report on the computational design of a distinctive class of biohybrid active membrane layer, built from the directed-insertion of aquaporin-incorporated lipid shell in to a silica nanopore. First, we describe in detail the mechanisms at play in the insertion of the biological membrane in to the solid-state nanopore. Then we analyze the structural stability of the system and demonstrate that its water permeability is comparable to the one measured in the biological environment. Finally, we discuss how the technology implemented could be applicable to environmental and biomedical applications, such as water desalination and drug discovery, where targeting and controlled permeation of small molecules must be efficiently addressed.

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“…Target proteins used in screening assays can be expressed in E. coli or in eukaryotic cells. The former has a much higher yield and cost efficiency; however, this is challenging when membrane proteins are needed [2,3]. The latter requires time-and reagent-consuming handling and provides limited control of membrane insertion and accessibility of the target protein.…”

Section: Introductionmentioning

confidence: 99%

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

et al. 2021

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Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the E. coli membrane. In order to make this channel accessible for the soluble compounds, E. coli were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay.

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Membrane Protein Stabilization Strategies for Structural and Functional Studies

Cited by 26 publications

References 139 publications

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Biohybrid membrane formation by directed insertion of Aquaporin into a solid-state nanopore

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

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