Optimizing Transmembrane Protein Assemblies in Nanodiscs for Structural Studies: A Comprehensive Manual
Fernando Vilela,
Cécile Sauvanet,
Armel Bezault,
Niels Volkmann,
Dorit Hanein
Affiliations
Fernando Vilela
Structural Studies of Macromolecular Machines in Cellulo Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR3528, Paris, FranceStructural Image Analysis Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR3528, Paris, France
Cécile Sauvanet
Structural Studies of Macromolecular Machines in Cellulo Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR3528, Paris, FranceStructural Image Analysis Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR3528, Paris, France
Armel Bezault
Structural Studies of Macromolecular Machines in Cellulo Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR3528, Paris, FranceStructural Image Analysis Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR3528, Paris, France
Niels Volkmann
Structural Image Analysis Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR3528, Paris, FranceDepartment of Biological Engineering; Department of Electrical and Computer Engineering University of California, Santa Barbara, CA, USA
Dorit Hanein
Department of Chemistry and Biochemistry, Department of Biological Engineering, University of California, Santa Barbara, CA, USA
Membrane protein structures offer a more accurate basis for understanding their functional correlates when derived from full-length proteins in their native lipid environment. Producing such samples has been a primary challenge in the field. Here, we present robust, step-by-step biochemical and biophysical protocols for generating monodisperse assemblies of full-length transmembrane proteins within lipidic environments. These protocols are particularly tailored for cases where the size and molecular weight of the proteins align closely with those of the lipid islands (nanodiscs). While designed for single-span bitopic membrane proteins, these protocols can be easily extended to proteins with multiple transmembrane domains. The insights presented have broad implications across diverse fields, including biophysics, structural biology, and cryogenic electron microscopy (cryo-EM) studies.
