1. 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
- Author
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Søren Feddersen, Jens K. Villadsen, Majken Wadum, Thomas B. F. Neergaard, Rikke S. Møller, Jens Knudsen, Peter Højrup, Nils J. Færgeman, and Birthe B. Kragelund
- Subjects
Models, Molecular ,Stereochemistry ,In Vitro Techniques ,Fatty Acids, Nonesterified ,Ligands ,Biochemistry ,Acyl-CoA ,chemistry.chemical_compound ,Protein structure ,Coenzyme A Ligases ,Acyl-CoA-binding protein ,Animals ,Molecular Biology ,Fluorescent Dyes ,Diazepam Binding Inhibitor ,chemistry.chemical_classification ,Base Sequence ,Esterification ,Escherichia coli Proteins ,Fatty acid ,DNA ,Cell Biology ,Metabolism ,Fluorescence ,Recombinant Proteins ,Protein Structure, Tertiary ,Kinetics ,chemistry ,Yield (chemistry) ,Mutagenesis, Site-Directed ,Cattle ,lipids (amino acids, peptides, and proteins) ,Acyl Coenzyme A ,Research Article ,Cysteine - Abstract
Long-chain acyl-CoA esters are key metabolites in lipid synthesis and β-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 525nm respectively, in the absence of ligand (excitation 387nm). 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 495nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460nm 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 (Kd = 0.6–1.7nM). 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.
- Published
- 2002