Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets |
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Authors: | Yann Ferrandez Manuela Dezi Mickael Bosco Agathe Urvoas Marie Valerio-Lepiniec Christel Le Bon Fabrice Giusti Isabelle Broutin Grégory Durand Ange Polidori Jean-Luc Popot Martin Picard Philippe Minard |
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Affiliation: | 1. Laboratoire de Modélisation et Ingénierie des Protéines, IBBMC UMR 8619, CNRS/Université Paris Sud, 91405, Orsay, France 2. Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie, UMR 8015, CNRS/Université Paris Descartes, Sorbonne Paris Cité, 4 avenue de l’Observatoire, 75270, Paris Cedex 06, France 3. Equipe Chimie Bioorganique et Systèmes Amphiphiles, Université d’Avignon, 33 rue Louis Pasteur, 84000, Avignon, France 4. Institut des Biomolécules Max Mousseron (UMR 5247), 15 avenue Charles Flahault, 34093, Montpellier Cedex 05, France 5. Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, Institut de Biologie Physico-Chimique (FRC 550), Centre National de la Recherche Scientifique/Université Paris-7, 13, rue Pierre-et-Marie-Curie, 75005, Paris, France
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Abstract: | Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for “artificial alpha repeat protein”) have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal “Velcro” to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself. |
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