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 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.
Wadum
Majken C T
MC
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Villadsen
Jens K
JK
Feddersen
Søren
S
Møller
Rikke S
RS
Neergaard
Thomas B F
TB
Kragelund
Birthe B
BB
Højrup
Peter
P
Faergeman
Nils J
NJ
Knudsen
Jens
J
D000214:Acyl Coenzyme A / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003066:Coenzyme A Ligases / Q000032:analysis; D004247:DNA / Q000235:genetics; D026261:Diazepam Binding Inhibitor / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis; D004951:Esterification; D005230:Fatty Acids, Nonesterified / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000214:Acyl Coenzyme A; D026261:Diazepam Binding Inhibitor; D029968:Escherichia coli Proteins; D005230:Fatty Acids, Nonesterified; D005456:Fluorescent Dyes; D008024:Ligands; D011994:Recombinant Proteins; D004247:DNA; D003066:Coenzyme A Ligases; C085934:acyl-coenzyme A synthetase, E coli