Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions
Huaying Zhao,
Yan Fu,
Carla Glasser,
Eric J Andrade Alba,
Mark L Mayer,
George Patterson,
Peter Schuck
Affiliations
Huaying Zhao
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
Yan Fu
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States
Carla Glasser
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
Eric J Andrade Alba
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States
Mark L Mayer
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
George Patterson
Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, United States
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
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.
