Abstract
The aryl hydrocarbon receptor nuclear transporter (ARNT) is a basic helix-loop-helix (bHLH) protein that contains a Per-Arnt-Sim (PAS) domain. ARNT heterodimerizes in vivo with other bHLH PAS proteins to regulate a number of cellular activities, but a physiological role for ARNT homodimers has not yet been established. Moreover, no rigorous studies have been done to characterize the biochemical properties of the bHLH domain of ARNT that would address this issue. To begin this characterization, we chemically synthesized a 56-residue peptide encompassing the bHLH domain of ARNT (residues 90-145). In the absence of DNA, the ARNT-bHLH peptide can form homodimers in lower ionic strength, as evidenced by dynamic light scattering analysis, and can bind E-box DNA (CACGTG) with high specificity and affinity, as determined by fluorescence anisotropy. Dimers and tetramers of ARNT-bHLH are observed bound to DNA in equilibrium sedimentation and dynamic light scattering experiments. The homodimeric peptide also undergoes a coil-to-helix transition upon E-box DNA binding. Peptide oligomerization and DNA affinity are strongly influenced by ionic strength. These biochemical and biophysical studies on the ARNT-bHLH reveal its inherent ability to form homodimers at concentrations supporting a physiological function and underscore the significant biochemical differences among the bHLH superfamily.
Highlights
Erodimerizes in vivo with other basic helix-loop-helix (bHLH) PAS proteins to regulate a number of cellular activities, but a physiological role for aryl hydrocarbon receptor nuclear transporter (ARNT) homodimers has not yet been established
ARNT heterodimerizes in vivo with other bHLH PAS proteins, including the aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor 1␣ (HIF1␣), to form activated DNA binding complexes (11)
Polypeptide Synthesis and Circular Dichroism—We synthesized a 56-aminoacyl residue polypeptide that corresponds to the bHLH DNA binding domain of the ARNT protein as determined by amino acid sequence homology with other bHLH proteins (Refs. 5–10, Fig. 1)
Summary
A peptide encompassing residues 90 –145 of ARNT (ARNT-bHLH) was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a Milligen/Biosearch peptide synthesizer. The CD spectra of both 100 M ARNT-bHLH peptide, calculated for a monomer, and 50 M duplex ARNDNA in Buffer A (50 mM NaCl or 50 mM NaF, 20 mM Tris, pH 7.4) were measured from 260 to 180 nm at 20 °C in a 0.01-cm cell. PeptideDNA experiments were performed by mixing 1.56 mM ARNT-bHLH peptide with 1.0 mM oligonucleotide (in 10 mM sodium cacodylate, pH 6.5), resulting in final concentrations of 0.78 and 0.50 mM each molecule, respectively. Under these experimental conditions, there is stoichiometric binding (data not shown). The value MA represents the average molecular weight of all sedimenting species. rM is the distance from the meniscus to the axis of the rotor, r is the distance between each point along the concentration gradient and the rotor axis, and C0 and CM are the concentrations at points r and rM, respectively. , , , (1 Ϫ ), R, and T represent angular velocity, partial specific volume, density, buoyancy, gas constant, and temperature, respectively
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