Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs

Bernhard, Max; Laube, Bodo

doi:10.1038/s41598-020-73157-2

Cited by 8 publications

(3 citation statements)

References 47 publications

(65 reference statements)

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“…A variety of methods have been developed to study the binding of ligands to macromolecules, including several that do not make use of labeled ligands, such as isothermal-titration calorimetry (e.g., Wöhri et al, 2013 ), surface plasmon resonance (e.g., Seeger et al, 2012 ), and microscale thermophoresis (e.g., Bernhard and Laube, 2020 ). One of the key advantages of methods that do use labeled ligands, however, is that the relationship between the observed signal and the ligand-binding phenomenon under study is most straightforward.…”

Section: Resultsmentioning

confidence: 99%

Probing function in ligand-gated ion channels without measuring ion transport

Godellas

Grosman

2022

Journal of General Physiology

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Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel’s affinities for ligands that bind to the extracellular domain.

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Section: Resultsmentioning

confidence: 99%

Probing function in ligand-gated ion channels without measuring ion transport

Godellas

Grosman

2022

Journal of General Physiology

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“…MST measurements showed that the full agonist glycine and the partial agonist taurine induced different conformational transitions on binding to the GlyR, giving EC50 values of 65 ± 22.8 µM and 473.8 ± 66.1 µM, respectively (Figure 4). The results indicated that partial agonism in pLGIC proteins is reflected by the adaption of distinct receptor conformations [49]. (a) Dose-response data for glycine and taurine of heterologous expressed α1 GlyR from Xenopus laevis oocytes.…”

Section: Other Receptorsmentioning

confidence: 99%

Microscale Thermophoresis Analysis of Membrane Proteins

Patching

2024

Preprint

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Microscale thermophoresis (MST) is an analytical technique for measuring biomolecular interactions. It is based on the physical phenomenon that particles move within temperature gradients, which is affected by their size, charge, hydration shell and conformation. The MST sample must contain a fluorescent target molecule used to observe the movement of particles, and this can be titrated with an unlabelled binding partner for quantifying the interaction. MST is highly sensitive, using relatively small amounts of sample, and it has no limitations on the size of the target biomolecule, on the affinity of the interaction or on the composition of the buffer and other sample components. This makes MST ideally suited to characterising interactions with membrane proteins, which can be studied in cell lysates, native membranes, solubilised in detergents or reconstituted in lipids. The intrinsic aromatic residues of membrane proteins have been used as the fluorophore for MST (label-free MST) or membrane proteins have been labelled with a range of fluorescent dyes or conjugated with fluorescent proteins (labelled MST). The different types of membrane proteins that have had biomolecular interactions characterised by MST include the SARS-CoV-2 spike protein, GPCRs and other receptors, sensor kinases, ion channels, aquaporins, and transport proteins.

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“…33 The different systems were chosen to investigate both the suitability of different lipid models to the application of MST and lipid-AMP binding, and the impact the choice of model system can have on the binding. SMA nanodiscs and vesicles were chosen as they have been previously utilised in MST, 23,34 and the effects of differences in surface curvature could be investigated. SMA-QA was selected as it possesses a cationic polymer belt, in contrast to SMA which has an anionic belt.…”

Section: Introductionmentioning

confidence: 99%

Label-free measurement of antimicrobial peptide interactions with lipid vesicles and nanodiscs using microscale thermophoresis

Isaksson

Rainsford

Rylandsholm

et al. 2023

Preprint

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One strategy to combat antimicrobial resistance is the discovery of new classes of antibiotics. Most antibiotics will at some point interact with the bacterial membrane to either interfere with its integrity or to cross it. Reliable and efficient tools for determining the dissociation constant for membrane binding (KD) and the partitioning coefficient between the aqueous- and membrane phases (KP) are therefore important tools for discovering and optimizing antimicrobial hits. Here we demonstrate that microscale thermophoresis (MST) can be used for label-free measurement of KD by utilising the intrinsic fluorescence of tryptophan and thereby removing the need for chromophore labelling. As proof of principle, we have used the method to measure the binding of a set of small cyclic AMPs to large unilamellar vesicles (LUVs) and two types of lipid nanodiscs assembled by styrene maleic acid (SMA) and quaternary ammonium SMA (SMA-QA). The measured KD values correlate well with the corresponding measurements using surface plasmon resonance (SPR), also broadly reflecting the tested AMPs’ minimal inhibition concentration (MIC) towards S. aureus and E. coli. We conclude that MST is a promising method for fast and cost-efficient detection of peptide-lipid interactions.

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Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs

Cited by 8 publications

References 47 publications

Probing function in ligand-gated ion channels without measuring ion transport

Probing function in ligand-gated ion channels without measuring ion transport

Microscale Thermophoresis Analysis of Membrane Proteins

Label-free measurement of antimicrobial peptide interactions with lipid vesicles and nanodiscs using microscale thermophoresis

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