Fig. 1. Mouse recombinant his-ACBP cloning and purification. (A) Agarose gels of the his-ACBP PCR product, his-ACBP PCR fragment cloned into pGEM-T vector, and his-ACBP subcloned into pET21b vector: HMW, high molecular weight marker (1 kb); LMW, low molecular weight marker (100 bp); H-ACBP-PCR, PCR product encoding his-ACBP cDNA; pGEM-H-ACBP, his-ACBP PCR fragment T-A cloned into the pGEM-T plasmid: 1, 1 μg of uncut plasmid; 2, 1 μg of XhoI/NdeI cut plasmid; pET21b-H-ACBP refers to his-ACBP cDNA cloned into pET21b plasmid: 3, 1 μg of XhoI-cut plasmid; 4, 1 μg of XhoI/NdeI digested plasmid. (B) DNA sequence of mouse ACBP cDNA inserted into pET21b vector and its amino acid translation. (C) SDS–PAGE gel and Western blotting of his-ACBP expressed in BL21 E. coli cells in the absence (lanes 1 and 3, respectively) and presence of IPTG (lanes 2 and 4, respectively). For SDS–PAGE and Coomassie staining, 20 μg of total protein was loaded per lane, while 10 μg of total protein per lane was used for Western blotting. (D) Representative Coomassie stained SDS–PAGE gel showing his-ACBP in E. coli cell lysate (lane 1, 25 μg protein) and elution fraction after Ni-column (lane 2, 2 μg protein). SDS–PAGE gel of purified his-ACBP silver stained to determine purity: 3, protein marker; 4, ultra low range protein marker; 5, 1 μg of his-ACBP; 6, 2 μg of his-ACBP; 7, 5 μg of his-ACBP.

Fig. 2. Structural characteristics of his-ACBP versus untagged ACBP: UV absorbance, CD and fluorescence spectra. (A) UV–vis spectra of 5 μM his-ACBP (solid) and 5 μM untagged ACBP (dashed). (B) Far UV CD spectra of his-tagged ACBP (solid) and ACBP (open). All CD studies were performed with 4 μM protein. (C) Fluorescence emission spectra of 200 nM his-ACBP (solid) and 200 nM ACBP (dashed) with excitation at 280 nm. (D) Fluorescence emission spectra of 200 nM his-ACBP (solid) and 200 nM ACBP (dashed) with excitation at 295 nm.

Fig. 3. Affinity and number of binding sites of his-ACBP with cis-parinaroyl-CoA (cPNCoA) and cis-parinaric acid (cPNA). Scatter plot and curve fitting of maximal fluorescence intensity (ex 310 nm, em 410 nm) with increasing of cPNCoA (A) and cPNA (A, inset) in presence of 30 nM his-ACBP. Reverse titrations of constant amounts (50 nM) of cPNCoA (B) and cPNA (B, inset) with increasing concentrations of his-ACBP indicated saturation at a molar ratio of 1 with cPNCoA, and non-specific binding for cPNA. (C) UV spectra of cPNCoA (4 μM) in potassium phosphate buffer, pH 7.4 (solid), cPNCoA spectra with increasing his-ACBP (0.4–16 μM) are represented by dashed and dotted lines. (D) Ratios of absorbance of valley 1 (V1)/peak 2 (P2) from spectra in (C) plotted versus protein/ligand molar ratio. (E) Wavelengths of peak 2 (P2) in (C) plotted versus protein/ligand molar ratio.

Fig. 4. Structural and functional characterization of Cy5-labeled his-ACBP. (A) SDS–PAGE of Cy5-his-ACBP and his-ACBP. (B) Western blotting of Cy5-his-ACBP and his-ACBP, 500 ng of protein per lane. (C) Far UV CD spectrum of his-ACBP and Cy5-his-ACBP. All CD studies were performed with 4 μM protein. (D) Titration and binding curve of Cy5-his-ACBP with cis-parinaroyl-CoA.

Fig. 5. Interaction of his-ACBP with HNF-4α by FRET. Cy3-HNF-4α (200 nM) and Cy3-β-galactosidase (200 nM, negative control) were titrated with increasing Cy5-his-ACBP (5 nM–2 μM) as described in Methods. (A) Emission spectra of Cy3-HNF-4α/Cy5-his-ACBP with donor Cy3 excitation at 500 nm; inset, quenching of donor Cy3 fluorescence versus acceptor Cy5-his-ACBP concentrations. (B) Close-up of spectra in (A), 640–750 nm range; inset, sensitized emission intensity for acceptor Cy5-his-ACBP versus Cy5-his-ACBP concentration. (C) Emission spectra of Cy3-β-galactosidase excited at 500 nm; inset, quenching in Cy3 versus Cy5-his-ACBP concentrations.

Binding affinity and FRET parameters for interaction between Cy3-HNF-4α and Cy5-his-ACBP or Cy5-ACBP

All parameters were calculated from FRET curves as presented in Fig. 5 (Methods). K_d, dissociation constant in nM; R_2/3, actual distance between Cy3 donor molecules on HNF-4α and Cy5 acceptor molecules on ACBP, in angstroms. SD values from n = 3.

Effect of his-tagging on ACBP enhancement of microsomal glycerol-3-phosphate acyltransferase (GPAT) and acyl CoA synthetase (ACS)

ND, not determined. Values represent means ± SE, n = 3 or 4.