title: Luminescence resonance energy transfer to study conformational changes in membrane proteins expressed in mammalian cells.
abstract: Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 Å. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston.
authors.forename: Drew M
authors.initials: DM
authors.lastname: Dolino
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston.
authors.forename: Swarna S
authors.initials: SS
authors.lastname: Ramaswamy
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston; Vasanthi.Jayaraman@uth.tmc.edu.
authors.forename: Vasanthi
authors.initials: V
authors.lastname: Jayaraman
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D018091:Receptors, AMPA / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D008565:Membrane Proteins; D018091:Receptors, AMPA