Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids

Majken C T Wadum, Jens Villadsen, Søren Feddersen, Rikke Steensbjerre Møller, Thomas B F Neergaard, Birthe Brandt Kragelund, Peter Højrup, Nils J. Færgeman, Jens Knudsen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.
Original languageEnglish
JournalBiochemical Journal
Volume365
Issue numberPt 1
Pages (from-to)165-72
ISSN0264-6021
DOIs
Publication statusPublished - 1. Jul 2002

    Fingerprint

Keywords

  • Acyl Coenzyme A
  • Animals
  • Base Sequence
  • Cattle
  • Coenzyme A Ligases
  • DNA
  • Diazepam Binding Inhibitor
  • Escherichia coli Proteins
  • Esterification
  • Fatty Acids, Nonesterified
  • Fluorescent Dyes
  • Kinetics
  • Ligands
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Recombinant Proteins

Cite this