Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse.
Acyl coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his-tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally occurring fluorescent cis-parinaroyl-CoA with very high affinity (K(d)=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his-tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor-4alpha (HNF-4alpha), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4alpha were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4alpha (intermolecular distance of 73 A) at high affinity (K(d)=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP.
Petrescu
Anca D
AD
Department of Physiology and Pharmacology, Texas A&M University, 4467 TAMU, TVMC, College Station, TX 77843-4467, USA.
Huang
Huan
H
Hostetler
Heather A
HA
Schroeder
Friedhelm
F
Kier
Ann B
AB
D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002232:Carbocyanines / Q000737:chemistry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000378:metabolism; D026261:Diazepam Binding Inhibitor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism*; D051557:Hepatocyte Nuclear Factor 4 / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000214:Acyl Coenzyme A; D002232:Carbocyanines; D026261:Diazepam Binding Inhibitor; D005231:Fatty Acids, Unsaturated; D051557:Hepatocyte Nuclear Factor 4; D011994:Recombinant Proteins; C089187:cyanine dye 3; C085321:cyanine dye 5; C086427:parinaroyl-coenzyme A; D006639:Histidine; D005992:Glycerol-3-Phosphate O-Acyltransferase; D003066:Coenzyme A Ligases; C013641:parinaric acid