Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents

ACS Chem Biol. 2021 Aug 20;16(8):1493-1507. doi: 10.1021/acschembio.1c00364. Epub 2021 Aug 6.

Abstract

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkynes / chemical synthesis
  • Alkynes / chemistry
  • Alkynes / metabolism
  • Binding Sites
  • Cholesterol / analogs & derivatives*
  • Cholesterol / chemical synthesis
  • Cholesterol / metabolism
  • Cyanobacteria / chemistry
  • Diazomethane / analogs & derivatives*
  • Diazomethane / chemical synthesis
  • Diazomethane / metabolism
  • Fluorescent Dyes / chemistry
  • Ligand-Gated Ion Channels / chemistry*
  • Ligand-Gated Ion Channels / metabolism
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Photoaffinity Labels / chemical synthesis
  • Photoaffinity Labels / chemistry*
  • Photoaffinity Labels / metabolism
  • Protein Binding

Substances

  • Alkynes
  • Fluorescent Dyes
  • Ligand-Gated Ion Channels
  • Photoaffinity Labels
  • Diazomethane
  • Cholesterol