TY - JOUR TI - Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions AU - Zhao, Huaying AU - Fu, Yan AU - Glasser, Carla AU - Andrade Alba, Eric J AU - Mayer, Mark L AU - Patterson, George AU - Schuck, Peter A2 - van Oijen, Antoine M VL - 5 PY - 2016 DA - 2016/07/20 SP - e17812 C1 - eLife 2016;5:e17812 DO - 10.7554/eLife.17812 UR - https://doi.org/10.7554/eLife.17812 AB - 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. KW - protein interactions KW - photoswitchable fluorescent proteins KW - analytical ultracentrifugation KW - hydrodynamics JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -