Structural effects of extracellular loop mutations in CFTR helical hairpins

Chang, Yuan-Heng; Stone, Tracy A.; Chin, Stephanie; Glibowicka, Mira; Bear, Christine E.; Deber, Charles M.

doi:10.1016/j.bbamem.2018.01.003

Cited by 5 publications

(3 citation statements)

References 36 publications

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“…This, in turn, may influence the negative impact of the V232D mutation on misfolding, for example, by enabling the D232 locus in mutant CFTR to favorably interact with neighboring, positively charged residues during biogenesis to stabilize, at least partially, a topically correct fold. Such a scenario could explain the mild form of cystic fibrosis caused by the V232D mutation 30 , in which maturation is not completely abolished but levels at ~20% of normal CFTR maturation 11 , 31 .…”

Section: Discussionmentioning

confidence: 99%

A minimal helical-hairpin motif provides molecular-level insights into misfolding and pharmacological rescue of CFTR

et al. 2018

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Our meagre understanding of CFTR misfolding and its reversal by small-molecule correctors hampers the development of mechanism-based therapies of cystic fibrosis. Here we exploit a helical-hairpin construct—the simplest proxy of membrane-protein tertiary contacts—containing CFTR’s transmembrane helices 3 and 4 and its corresponding disease phenotypic mutant V232D to gain molecular-level insights into CFTR misfolding and drug rescue by the corrector Lumacaftor. Using a single-molecule FRET approach to study hairpin conformations in lipid bilayers, we find that the wild-type hairpin is well folded, whereas the V232D mutant assumes an open conformation in bilayer thicknesses mimicking the endoplasmic reticulum. Addition of Lumacaftor reverses the aberrant opening of the mutant hairpin to restore a compact state as in the wild type. The observed membrane escape of the V232D hairpin and its reversal by Lumacaftor complement cell-based analyses of the full-length protein, thereby providing in vivo and in vitro correlates of CFTR misfolding and drug-action mechanisms.

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Section: Discussionmentioning

confidence: 99%

A minimal helical-hairpin motif provides molecular-level insights into misfolding and pharmacological rescue of CFTR

et al. 2018

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“…4a, b and Supplementary Table 4 ). All three mutations are CF-phenotypic and known to cause maturation defects and misfolding of CFTR 63 , 64 . The drug molecules were two CFTR interacting correctors, Lumacaftor (VX-809) and Galicaftor (ABBV-2222) 65 .…”

Section: Resultsmentioning

confidence: 99%

An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Hartmann,

Sreenivasa,

Schenkel

et al. 2023

Nat Commun

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Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein–DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.

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“…S4). All three mutations are CF-phenotypic and known to cause maturation defects and misfolding of CFTR 55,56 . The drug molecules were two CFTR interacting correctors, Lumacaftor (VX-809) and Galicaftor (ABBV-2222) 57 .…”

Section: Multiwell Plate Smfret Screening Of Drug-protein Interactionsmentioning

confidence: 99%

Farewell to single-well: An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Hartmann

Sreenivasa

Schenkel

et al. 2023

Preprint

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Single-molecule FRET (smFRET) has become a widely used tool for probing the structure, dynamics, and functional mechanisms of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Investigations by smFRET often require sampling of a large parameter space, for example, by varying one or more constituent molecular components in ten or more steps to reliably extract distances, kinetic rates, and other quantitative parameters. Confocal smFRET measurements, for example, which are amongst the widely used smFRET assays, are typically performed in a single-well format and measurements are conducted in a manual manner, making sampling of many experimental parameters laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality into a confocal microscope to allow for continuous and automated smFRET measurements. We show that the multiwell plate assay is on par with conventional single-well smFRET measurements in terms of accuracy and precision yet enables probing tens to hundreds of conditions in a fully automized manner. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, and competitive and cooperative protein–DNA interactions, revealing new insights that would be hard if not impossible to achieve with conventional single-well format measurements. The higher sampling density afforded by the multiwell plate format increases the accuracy of data analysis by at least 10-fold. We further showcase that the assay provides access to smFRET-based screening of drug–protein interactions. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software. Taken together, the automated multiwell plate assay developed here opens up new possibilities to acquire high-content smFRET datasets for in-depth single-molecule analysis of biomolecular conformations, interactions, and dynamics.

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Structural effects of extracellular loop mutations in CFTR helical hairpins

Cited by 5 publications

References 36 publications

A minimal helical-hairpin motif provides molecular-level insights into misfolding and pharmacological rescue of CFTR

A minimal helical-hairpin motif provides molecular-level insights into misfolding and pharmacological rescue of CFTR

An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Farewell to single-well: An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

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