High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy

Aaron J. Wolfe, Kyle J. Parella, Liviu Movileanu

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)e96
JournalCurrent protocols in protein science
Volume97
Issue number1
DOIs
StatePublished - Sep 1 2019

Fingerprint

Fluorescence Polarization
Fluorescence Spectrometry
Detergents
Screening
Fluorescence
Throughput
Spectroscopy
Polarization
Membrane Proteins
Proteins
Anisotropy
Rate constants
Stabilization
Association reactions
Kinetics
Research

Keywords

  • fluorescent labeling
  • kinetics
  • membrane protein engineering
  • protein folding
  • protein-detergent interactions
  • proteomicelles

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry

Cite this

High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy. / Wolfe, Aaron J.; Parella, Kyle J.; Movileanu, Liviu.

In: Current protocols in protein science, Vol. 97, No. 1, 01.09.2019, p. e96.

Research output: Contribution to journalArticle

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