Valium Research Today is a free monthly online journal that collates and summarizes the latest research about Valium, including details on diazepam, depression, side-effects, withdrawal. | ||||||||
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Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse.Petrescu AD, Huang H, Hostetler HA, Schroeder F, Kier AB Department of Physiology and Pharmacology, Texas A&M University, 4467 TAMU, TVMC, College Station, TX 77843-4467, USA. 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. Published 27 February 2008 in Protein Expr Purif, 58(2): 184-93.
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