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Surveying GPCR solubilisation conditions using surface plasmon resonance.

Iva Hopkins Navratilova1, Tonia Aristotelous2, Louise E Bird3

  • 1University of Dundee, Dow Street, Dundee, DD1 5EH, UK; Research Complex at Harwell, Rutherford Appleton Lab, Harwell, Oxford, OX11 0FA, UK.

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Optimizing buffer and detergent conditions is key for studying solubilized G-protein coupled receptors (GPCRs). This study screened 950 conditions for CXCR4 and CCR5, revealing specific preferences for maintaining receptor activity and assay sensitivity.

Keywords:
DetergentsGPCRsScreeningSolubilisation conditionsSurface plasmon resonance

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • Solubilized G-protein coupled receptors (GPCRs) require specific environmental conditions for activity.
  • Determining optimal solubilization buffers is challenging due to numerous variables.
  • Biophysical screening methods like surface plasmon resonance (SPR) are vital for kinetic analysis.

Purpose of the Study:

  • To screen and identify optimal solubilization conditions (buffers and detergents) for CXCR4 and CCR5.
  • To investigate factors influencing assay sensitivity for these receptors.
  • To provide guidance for solubilizing other membrane proteins.

Main Methods:

  • Utilized surface plasmon resonance (SPR) technology for biophysical screening.
  • Tested 950 different combinations of buffers and detergents for CXCR4 and CCR5.
  • Monitored receptor activity using conformation-dependent monoclonal antibodies and small molecule ligand binding.

Main Results:

  • Identified distinct optimal solubilization conditions for CXCR4 and CCR5.
  • Observed differences in receptor preferences for buffers and detergents, despite belonging to the same family.
  • Found that optimal conditions enhanced binding of both antibodies and small molecules, impacting assay sensitivity.

Conclusions:

  • Solubilization buffer and detergent composition significantly impacts GPCR activity and SPR assay sensitivity.
  • Receptor-specific optimization is necessary, even within the same protein family.
  • The presented data can serve as a valuable guide for solubilizing diverse membrane proteins.