PROTOCOL
High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy
Aaron J. Wolfe,
Kyle J. Parella,
Liviu Movileanu,
Aaron J. Wolfe
Ichor Therapeutics, Inc., LaFayette, New York
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York
Search for more papers by this authorKyle J. Parella
Ichor Therapeutics, Inc., LaFayette, New York
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York
Search for more papers by this authorCorresponding Author
Liviu Movileanu
Department of Physics, Syracuse University, Syracuse, New York
Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York
Corresponding author: [email protected]Search for more papers by this authorAaron J. Wolfe,
Kyle J. Parella,
Liviu Movileanu,
Aaron J. Wolfe
Ichor Therapeutics, Inc., LaFayette, New York
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York
Search for more papers by this authorKyle J. Parella
Ichor Therapeutics, Inc., LaFayette, New York
Department of Chemistry, State University of New York College of Environmental Science and Forestry, Syracuse, New York
Search for more papers by this authorCorresponding Author
Liviu Movileanu
Department of Physics, Syracuse University, Syracuse, New York
Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York
Corresponding author: [email protected]Search for more papers by this authorFirst published: 30 August 2019
Abstract
This article provides detailed protocols for a high-throughput fluorescence polarization (FP) spectroscopy approach to disentangle the interactions of membrane proteins with solubilizing detergents. Existing techniques for examining the membrane protein-detergent complex (PDC) interactions are low throughput and require high amounts of proteins. Here, we describe a 96-well analytical approach, which facilitates a scalable analysis of the PDC interactions at low-nanomolar concentrations of membrane proteins in native solutions. At detergent concentrations much greater than the equilibrium dissociation constant of the PDC, Kd, the FP anisotropy reaches a saturated value, so it is independent of the detergent concentration. On the contrary, at detergent concentrations comparable with or lower than the Kd, the FP anisotropy readout undergoes a time-dependent decrease, exhibiting a sensitive and specific detergent-dissociation signature. Our approach can also be used for determining the kinetic rate constants of association and dissociation. With further development, these protocols might be used in various arenas of membrane protein research that pertain to extraction, solubilization, and stabilization. © 2019 by John Wiley & Sons, Inc.
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