title: Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions.
abstract: The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors.
authors.affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States.
authors.forename: Huaying
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Carla
authors.initials: C
authors.lastname: Glasser
authors.affiliation: Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Andrade Alba
authors.affiliation: Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Mayer
authors.affiliation: Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States.
authors.forename: George
authors.initials: G
authors.lastname: Patterson
authors.affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States.
authors.forename: Peter
authors.identifier: 0000-0002-8859-6966
authors.initials: P
authors.lastname: Schuck
mesh_terms: D002623:Chemistry Techniques, Analytical; D005470:Fluorometry; D046912:Multiprotein Complexes / Q000737:chemistry*; D025941:Protein Interaction Mapping; D011506:Proteins / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods; D014461:Ultracentrifugation
chemical_list: D046912:Multiprotein Complexes; D011506:Proteins
keywords: analytical ultracentrifugation; biophysics; hydrodynamics; photoswitchable fluorescent proteins; protein interactions; structural biology