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
Long-chain acyl-CoA esters are key metabolites in lipid synthesis and b-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.
Biochemical Journal, 2002, Vol 365, Issue Pt 1, p. 165-172
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; 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, 9007-49-2 DNA, EC 6.2.1.- Coenzyme A Ligases, EC 6.2.1.- acyl-coenzyme A synthetase, E coli