B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles - Institut Pasteur Accéder directement au contenu
Pré-Publication, Document De Travail Année : 2023

B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles

Résumé

Amphitropic proteins and peptides reversibly partition from solution to membrane, a key process that regulates their functions. Experimental approaches, such as fluorescence and circular dichroism, are classically used to measure the partitioning of amphitropic peptides and proteins into lipid bilayers, yet hardly usable when the peptides or proteins do not exhibit significant polarity and/or conformational changes upon membrane binding. Here, we describe B2LiVe ( i . e ., Binding to Lipid Vesicles), a simple, robust, and widely applicable NMR method to determine the solution-to-membrane partitioning of unlabeled proteins or peptides. The experimental strategy proposed here relies on previously described proton 1D NMR fast pulsing techniques with selective adiabatic pulses. Membrane partitioning induces a large line broadening leading to a progressive loss of protein signals, and therefore, the decrease of the NMR signal directly measures the fraction of membrane-bound protein. The B2LiVe method uses low polypeptide concentrations and has been validated on several membrane-interacting peptides and proteins, ranging from 3 to 54 kDa, with membrane vesicles of different sizes and various lipid compositions. Motivation Characterization of the interaction of peptides and proteins with lipid membranes is involved in various biological processes and is often challenging for polypeptides which do not possess intrinsic fluorophores, do not exhibit significant structural content changes, as well as for those characterized by low affinities for membranes. To meet these challenges, we have developed a simple and robust label-free NMR-based experimental approach, named B2LiVe, to measure the binding of polypeptides to lipid vesicles. The experimental strategy relies on previously described proton 1D NMR fast pulsing techniques with selective adiabatic pulses to excite the amide resonances. B2LiVe is a label-free method based on the observation of amide hydrogen nuclei which are naturally present in all protein and peptide backbones. Our results validate the B2LiVe method and indicate that it compares well with established technics to quantify polypeptide-membrane interactions. Overall, B2LiVe should efficiently complement the arsenal of label-free biophysical assays available to characterize protein-membrane interactions. In brief We describe a robust label-free NMR-based experimental approach (B2LiVe) to measure interactions between peptides or proteins with membranes. The validity of this approach has been established on several polypeptides and on various membrane vesicles. The B2LiVe method efficiently complements the arsenal of label-free biophysical techniques to characterize protein-membrane interactions. Highlights B2LiVe is a simple and robust NMR-based method to quantify affinity of proteins and peptides for membranes B2LiVe is a label-free approach that relies on proton 1D NMR fast pulsing techniques with selective excitation of amide resonances B2LiVe has been validated on several membrane-interacting peptides and proteins B2LiVe coupled to DOSY can pinpoint the presence within a membrane-bound protein of polypeptide segments remaining in solution
Fichier principal
Vignette du fichier
2022.12.14.520471v1.full.pdf (1.67 Mo) Télécharger le fichier
Origine : Fichiers produits par l'(les) auteur(s)
Licence : CC BY NC ND - Paternité - Pas d'utilisation commerciale - Pas de modification

Dates et versions

pasteur-04356880 , version 1 (02-01-2023)
pasteur-04356880 , version 2 (20-12-2023)

Licence

Paternité - Pas d'utilisation commerciale - Pas de modification

Identifiants

Citer

Mirko Sadi, Nicolas Carvalho, Bruno Vitorge, Daniel Ladant, J.Inaki Guijarro, et al.. B2LiVe, a label-free 1D-NMR method to quantify the binding of amphitropic peptides or proteins to membrane vesicles. 2023. ⟨pasteur-04356880v1⟩
32 Consultations
30 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More