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• W2003075282 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 vivowith 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. 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 vivowith 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. aryl hydrocarbon receptor nuclear transporter basic helix-loop-helix Per-Arnt-Sim aryl hydrocarbon receptor hypoxia-inducible factor Deadpan circular dichroism trifluoroethanol The aryl hydrocarbon receptor nuclear transporter (ARNT)1 protein belongs to the basic-helix-loop-helix Per-Arnt-Sim (bHLH PAS) family of transcriptional regulator proteins. These functionally oligomeric proteins are important for cell cycle and developmental regulation and for sensing and responding to environmental conditions (1Zhulin I.B. Taylor B.L. Trends Biochem. Sci. 1997; 22: 331-333Abstract Full Text PDF PubMed Scopus (344) Google Scholar, 2Crews S.T. Genes Dev. 1998; 12: 607-620Crossref PubMed Scopus (301) Google Scholar). ARNT shows high sequence homology to other bHLH motifs of this family (3Antonsson C. Arulampalam V. Whitelaw M.L. Pettersson S. Poellinger L. J. Biol. Chem. 1995; 270: 13968-13972Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,4Basci S.G. Hankinson O. J. Biol. Chem. 1996; 271: 8843-8850Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), particularly at residues known to contact DNA (5Ferré-D'Amaré A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar, 6Ferré-D'Amaré A.R. Pognonec P. Roeder R.G. Burley S.K. EMBO J. 1994; 13: 180-189Crossref PubMed Scopus (333) Google Scholar, 7Ma P.C.M. Rould M.A. Weintraub H. Pabo C.O. Cell. 1994; 77: 451-459Abstract Full Text PDF PubMed Scopus (401) Google Scholar, 8Shimizu T. Toumoto A. Ihara K. Shimizu M. Kyogoku Y. Ogawa N. Oshima Y. Hakoshima T. EMBO J. 1997; 16: 4689-4697Crossref PubMed Scopus (155) Google Scholar, 9Ellenberger T.E. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Crossref PubMed Scopus (357) Google Scholar, 10Párraga A. Bellsolell L. Ferré-D'Amaré A.R. Burley S.K. Structure (Lond.). 1998; 6: 661-672Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In general, bHLH domains bind a consensus DNA element, the so-named E-box (CANNTG), and are required for oligomerization. PAS domains, which are found in all kingdoms, are involved in protein-protein interactions and ligand/inducer binding, acting as environmental sensors (1Zhulin I.B. Taylor B.L. Trends Biochem. Sci. 1997; 22: 331-333Abstract Full Text PDF PubMed Scopus (344) Google Scholar). PAS-containing proteins typically have two such conserved, repeated domains that are separated by a spacer region. PAS domains are not always contiguous with the bHLH DNA binding domain. ARNT heterodimerizes in vivo with other bHLH PAS proteins, including the aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor 1α (HIF1α), to form activated DNA binding complexes (11Gradin K. McGuire J. Wegner R.H. Kvietikova I. Whitelaw M.L. Toftgård R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Crossref PubMed Scopus (391) Google Scholar). Formation of the AHR/ARNT heterodimer requires the binding of polycyclic and halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, which are known exogenous ligands for AHR and mediate carcinogenesis via AHR·ARNT complex activation (reviewed in Ref. 12Hankinson O. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1438) Google Scholar). The resulting AHR·ARNT complex binds an atypical E-box DNA sequence, TNGCGTG, thereby activating the transcription of a number of target genes by direct interaction with general transcription factors, such as transcription factor IIB (13Swanson H.I. Yang J.H. Mol. Pharmacol. 1998; 54: 671-677PubMed Google Scholar). Among the activated genes are CYP1A1 and CYP1A2, cytochrome oxidases that metabolize polycyclic and aromatic compounds to electrophilic derivatives, which are the ultimate chemical agents that attack DNA. The AHR/ARNT heterodimer also activates transcription of the mdr1 multidrug transporter, albeit indirectly (14Mathieu M.-C. Lapierre I. Brault K. Raymond M. J. Biol. Chem. 2001; 276: 4819-4827Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The HIF1α/ARNT heterodimer (HIF1) senses the oxygen tension in cells and, under hypoxic conditions, activates the transcription of a number of genes, the promoters of which contain the E-box sequence, TACGTGCT. Transcription is activated by formation of a HIF1-(CREB (cAMP-response element-binding protein)/ATF1)-(p300/CBP (CREB-binding protein)) complex on the cognate DNA (15Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Crossref PubMed Scopus (232) Google Scholar). Among the genes regulated are erythropoietin, vascular endothelial growth factor, glycolytic enzymes, tyrosine hydroxylase, inducible nitric-oxide synthase, and heme oxygenase-1, all of which allow the cell to cope with lower oxygen levels (reviewed in Ref. 16Guillemin K. Krasnow M.A. Cell. 1997; 89: 9-12Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). HIF1 also plays a role in iron homeostasis by its activation of the ceruloplasm gene (17Mukhopadhyay C.K. Mazumder B. Fox P.L. J. Biol. Chem. 2000; 275: 21048-21054Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). This HIF1-mediated response has been found to be crucial for angiogenesis and solid tumor formation (18Ryan H.E. Lo J. Johnson R.S. EMBO J. 1998; 17: 3005-3015Crossref PubMed Scopus (1340) Google Scholar, 19Maxwell P.H. Dachs G.U. Gleadle J.M. Nicholls L.G. Harris A.L. Stratford I.J. Hankinson O. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8104-8109Crossref PubMed Scopus (951) Google Scholar, 20Carmeliet P. Dor Y. Herbert J.-M. Fukumura D. Brusselmans K. Derwerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshet E. Nature. 1998; 394: 485-490Crossref PubMed Scopus (2227) Google Scholar). Neither AHR or HIF1α homodimers nor AHR/HIF1α heterodimers have been observed. In contrast to the in vivo importance of AHR/ARNT and HIF1α/ARNT heterodimers, the biological relevance of ARNT homodimers is unclear. No physiological role for ARNT homodimers has yet been defined, and in vitro coimmunoprecipitation studies have been unable to detect the homodimeric ARNT complex (21Reisz-Porszasz S. Probst M.R. Fukunaga B.N. Hankinson O. Mol. Cell. Biol. 1994; 14: 6075-6086Crossref PubMed Scopus (217) Google Scholar). However,in vivo reporter gene assays have demonstrated that putative ARNT homodimers can activate transcription via E-box binding (3Antonsson C. Arulampalam V. Whitelaw M.L. Pettersson S. Poellinger L. J. Biol. Chem. 1995; 270: 13968-13972Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 22Sogawa K. Nakano R. Kobayashi A. Kikuchi Y. Ohe N. Matsushita N. Fujii-Kuriyama Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1936-1940Crossref PubMed Scopus (170) Google Scholar), and a preferred DNA binding site has been identified that contains the E-box sequence (23Swanson H.I. Yang J.-H. Nucleic Acids Res. 1999; 27: 3205-3212Crossref PubMed Scopus (39) Google Scholar). It is expected that ARNT homodimers would bind to the CACGTG E-box as do other canonical bHLH proteins, whereas ARNT in one of its heterodimeric complexes would bind one half-site of an asymmetric consensus binding site, with the heterodimeric partner binding the other, nonconsensus half-site. As demonstrated for the AHR/ARNT heterodimer, ARNT is located on the GTG E-box half-site, and AHR is situated on the less restrictive (A/C)(G/C/T)(A/T) non-E-box half-site (24Basci S.G. Reisz-Porszasz S. Hankinson O. Mol. Pharmacol. 1995; 47: 432-438PubMed Google Scholar). The structures of several bHLH domain-containing peptides bound to DNA have been determined by x-ray crystallography, including MAX, USF, MyoD, Pho4, E47, and SREBP1 (5Ferré-D'Amaré A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar, 6Ferré-D'Amaré A.R. Pognonec P. Roeder R.G. Burley S.K. EMBO J. 1994; 13: 180-189Crossref PubMed Scopus (333) Google Scholar, 7Ma P.C.M. Rould M.A. Weintraub H. Pabo C.O. Cell. 1994; 77: 451-459Abstract Full Text PDF PubMed Scopus (401) Google Scholar, 8Shimizu T. Toumoto A. Ihara K. Shimizu M. Kyogoku Y. Ogawa N. Oshima Y. Hakoshima T. EMBO J. 1997; 16: 4689-4697Crossref PubMed Scopus (155) Google Scholar, 9Ellenberger T.E. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Crossref PubMed Scopus (357) Google Scholar, 10Párraga A. Bellsolell L. Ferré-D'Amaré A.R. Burley S.K. Structure (Lond.). 1998; 6: 661-672Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). From these structures, it has been determined that the basic region is helical, and its residues make the primary contacts to the DNA, whereas the helix-loop-helix region is largely responsible for dimerization. Additionally, synthetic peptides of several bHLH domains have been characterized biochemically and biophysically, of which the Deadpan bHLH was perhaps the most rigorously investigated (25Wendt H. Thomas R.M. Ellenberger T. J. Biol. Chem. 1998; 273: 5735-5743Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 26Muhle-Goll C. Nilges M. Pastore A. Biochemistry. 1995; 34: 13554-13564Crossref PubMed Scopus (45) Google Scholar, 27Künne A.G.E. Allemann R.K. Biochemistry. 1997; 36: 1085-1091Crossref PubMed Scopus (12) Google Scholar, 28Anthony-Cahill S.J. Benfield P.A. Fairman R. Wasserman Z.R. Brenner S.L. Stafford III, W.F. Altenbach C. Hubbell W.L. DeGrado W.F. Science. 1992; 255: 979-983Crossref PubMed Scopus (139) Google Scholar, 29Winston R.L. Millar D.P. Gottesfeld J.M. Kent S.B. Biochemistry. 1999; 16: 5138-5146Crossref Scopus (28) Google Scholar, 30Bishop P. Ghosh I. Jones C. Chmielewski J. J. Am. Chem. Soc. 1995; 117: 8283-8284Crossref Scopus (10) Google Scholar). However, these latter studies do not address the biochemical and biophysical properties of the bHLH-PAS family members, in particular ARNT. To delineate the structural mechanism of transcription regulation by ARNT either as a homodimer or an AHR/ARNT or HIF1α/ARNT heterodimer, we have undertaken a series of biophysical and biochemical studies on a chemically synthesized, 56-residue peptide that encompasses the ARNT-bHLH domain. Specifically, we have used circular dichroism (CD) to determine the extent to which the peptide is folded in both the presence and absence of DNA and equilibrium sedimentation to determine its oligomerization state. Furthermore, using fluorescence anisotropy, we have determined the binding affinity of this peptide for E-box DNA under a variety of experimental conditions. As a complement to our sedimentation equilibrium experiments, we conducted dynamic light scattering studies to evaluate the oligomerization state and monodispersity of the peptide at higher concentrations. Unexpectedly, significant biochemical differences from other bHLH proteins such as Deadpan and Tal were found. Moreover, the data provide evidence that the bHLH of ARNT can form homodimers, which might have biological relevance. 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. Cleavage and deprotection reactions were carried out in trimethylsilane bromide, ethanediol, m-cresol, thioanisol, and trifluoroacetic acid for 1 h at 0 °C under a nitrogen blanket to ensure that cysteine residues remained reduced after removal of the trityl protection groups. The cleaved peptide was filtered through a medium-sintered glass filter to separate it from resin. The peptide was then washed with trifluoroacetic acid. The filtrate and washings were combined, and all liquid was evaporated using a rotary evaporator. The peptide was precipitated with diethyl ether and filtered through a medium-sintered glass funnel. The peptide was dried under a stream of nitrogen, dissolved in 10% acetic acid, and lyophilized. The peptide was then dissolved in 0.1% trifluoroacetic acid and purified on a Vydac C-18 reverse-phase HPLC column with a mobile phase of 0.1% trifluoroacetic acid and a linear 60-min gradient of 0–100% acetonitrile. Chromatography runs were recorded with a diode array detector and analyzed using Millennium 2000 software (Waters). The peptide eluted at 73% acetonitrile. Fractions from 70 to 74% were pooled and rechromatographed. The purity of the peptide was ascertained by mass spectrometry (data not shown). Circular dichroism experiments were performed with a Jasco-J500 instrument. An E-box-containing oligonucleotide, ARNDNA (5′-GGCTCAGTCACGTGACTGAGC-3′), was purchased from Oligos, Etc. This sequence was chosen to contain the consensus RTCACGTGAY sequence determined to be recognized by ARNT using a site affinity and amplification assay (23Swanson H.I. Yang J.-H. Nucleic Acids Res. 1999; 27: 3205-3212Crossref PubMed Scopus (39) Google Scholar). The oligonucleotide, which has an unpaired 5′ guanosine upon duplex formation, was resuspended from the lyophilized pellet in 10 mm sodium cacodylate, pH 6.5, such that the final concentration of single-stranded oligonucleotide was 2.0 mm. The concentration was calculated using data that were provided by Oligos, Etc. for each strand. The palindromic strands were annealed by heating to 80 °C, followed by slow cooling to 25 °C. Complete annealing was confirmed by high pressure liquid chromatography and gel electrophoresis (data not shown). The final concentration of duplex DNA was determined by measuring the absorbance of the solution at 260 nm and using the equation A = εcl, where A is the absorbance at 260 nm, ε is the known extinction coefficient for double-stranded DNA, 0.02 µg−1 cm−1 (31Sambrook J. Fritsch E.F. Maniatas T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: E5Google Scholar), c is the concentration of DNA in µg/ml, and l is the path length through the cuvette, 1 cm. The molecular weight of the duplex oligonucleotide (13.0 kDa) was subsequently used to calculate the molar concentration of ARNDNA. The CD spectra of both 100 µmARNT-bHLH peptide, calculated for a monomer, and 50 µmduplex ARNDNA in Buffer A (50 mm NaCl or 50 mmNaF, 20 mm Tris, pH 7.4) were measured from 260 to 180 nm at 20 °C in a 0.01-cm cell. The substitution of NaF for NaCl had no impact on the spectra (data for NaCl not shown). The spectrum of 100 µm ARNT containing 50% trifluoroethanol (TFE) or 50 µm duplex ARNDNA oligonucleotide was also measured. The final reported spectra are averages of 10 runs. To measure any changes in peptide secondary structure upon binding DNA, the CD difference spectrum was calculated by subtracting the spectrum of ARNDNA alone from that of the ARNDNA/ARNT mixture. The concentration of the peptide solution was verified by amino acid analysis. The secondary structures were analyzed using the variable selection method (32Compton L.A. Mathews C.K. Johnson W.C. J. Biol. Chem. 1987; 262: 13039-13043Abstract Full Text PDF PubMed Google Scholar). Fluorescence polarization experiments were done with a PanVera Beacon fluorescence polarization system (PanVera Corp.). 5′-Fluoresceinated oligonucleotides corresponding to ARNDNA and theEscherichia coli purF operator (Oligos, Etc.) (ARNDNA, 5′-F-GCTCAGTCACGTGACTGAGCCCCTTGCTCAGTCACGTGACTGAGC-3′,purF 5′-F-AAAGAAAACGTTTGCGTACCCCCTACGCAAACGTTTTCTTT-3′) were self-annealed in 10 mm sodium cacodylate, pH 6.5, by heating to 80 °C followed by flash cooling to form a stem-loop structure with the E-box motif (underlined) or purF operator motif, respectively, at the center of the stem. Oligonucleotide concentrations were calculated as described for ARNDNA used in CD experiments. Binding was assayed in a 1-ml volume at 25 °C. Unless otherwise noted in the text, the components of each binding experiment were 2 nm fluoresceinated DNA and 1.0 µg/ml poly(d[I·C]) in Buffer A (50 mm NaCl or NaF, 20 mm Tris, pH 7.4). Poly(d[I·C]) (Sigma) was included as a control for nonspecific DNA binding. It is expressed in µg/ml rather than molar to reflect the fact that the exact length of the poly(d[I·C]) molecules is not discrete but averages between 1200 and 3000 base pairs. In one set of experiments, the amount of poly(d[I·C]) included was varied to 0.0, 0.1, or 1.0 µg/ml. After each addition of peptide, samples were incubated in the Beacon instrument at 25 °C for 30 s before a measurement was taken. The 30-s incubation allowed equilibrium to be reached. The millipolarization (P ×10−3 where Pis polarization) at each titration point represents the average of eight measurements integrated over 6 s. Samples were excited at 490 nm, and emission was measured at 530 nm. The data of each binding isotherm were analyzed by curve fitting using SigmaPlot software (Jandel Corp.). Because the calculated dissociation constants were all greater than 20 nm and the experimental DNA concentration was 10-fold less than this value, it was assumed that the concentration of protein bound to DNA was negligible in comparison with the total protein concentration. Therefore, the following equation could be applied (33Lundblad J.R. Laurance M. Goodman R.H. Mol. Endocrinol. 1996; 10: 607-612Crossref PubMed Scopus (217) Google Scholar), P=((Pbound−Pfree)[ARNT]/(Kd+[ARNT]))+PfreeEquation 1 where P is the polarization measured at a given total concentration of peptide ([ARNT]), Pfree is the initial polarization of the free DNA, andPbound is the maximum polarization of specifically bound DNA. Nonlinear least squares analysis was used to determine Pfree, Pbound, and Kd. Anisotropy studies at higher salt concentrations (Buffer B: 150 mm NaCl, 100 mm Tris, pH 7.4) were done with an SLM 8000 spectrofluorometer with T optics at 25 °C. The sample was excited at 480 nm, and the parallel and perpendicular polarization components of fluorescein emission were measured at 520 nm. Except for the buffer, the components in the assay were the same as those used in measuring anisotropy in lower salt buffer. After each addition of peptide, the solution was incubated for 1 min to attain equilibrium. Each titration point is an average of 12 measurements, each integrated over 30 s. Data were analyzed using Scientist MicroMath software. Anisotropy was measured as the function of concentration of ARNT-bHLH (µm) added to the binding reaction, and the data were fit using a two-step binding model that follows. Anisotropy,A, is related to polarization, P, byA = 2P/(3 − P) (Ref.33Lundblad J.R. Laurance M. Goodman R.H. Mol. Endocrinol. 1996; 10: 607-612Crossref PubMed Scopus (217) Google Scholar). The equilibrium model that best describes the initial phase of the higher salt binding isotherm is two ARNT-bHLH monomers binding to the ARNDNA duplex, i.e. 2 ARNT-bHLH monomers + ARNDNA ↔ ARNT-bHLH·ARNDNA + ARNT-bHLH ↔ ARNT-bHLH dimer·ARNDNA. The equation to describe this cooperative binding is as follows, ν1=CP/CHALFP+CPEquation 2 where ν1 is the average number of bound ARNT-bHLH molecules per molecule of ARNDNA, C is the peptide concentration (µm), and CHALF is the concentration at half-saturation of the cooperative binding event.P, the Hill coefficient, is the average number of interacting sites on ARNDNA, which is two. P is also the measure of cooperativity in a binding event. The following equilibrium model describes the second phase of binding: 1 ARNT-bHLH dimer·ARNDNA + 1 ARNT-bHLH dimer ↔ 2 ARNT-bHLH dimers·ARNDNA. In noncooperative binding, all binding sites are equivalent and independent of each other. The binding curve is a rectangular hyperbola (34van Holde K.E. Physical Biochemistry. 2nd Ed. Prentice Hall, Upper Saddle River, NJ1985: 598-628Google Scholar) and is defined by the equation, ν2=K[ARNT]/(1+K[ARNT])Equation 3 where ν2 is the average number of ARNT-bHLH dimers bound to the ARNT-bHLH dimer·ARNDNA complex formed in the first step.K is the association constant of the noncooperative event, and [ARNT] is the concentration of free ARNT-bHLH monomers. At high concentrations (>23 µm), ARNT-bHLH is dimeric. In this step of binding, only the dimer-bound DNA is available for further binding of ARNT. Dynamic light scattering studies were done using a DynaPro-801 instrument (Protein Solutions, Inc.). All solutions were filtered through 0.1-µm Anotop filters (Whatman) to remove aggregated peptide and other particulates. Scattering of the ARNT-bHLH peptide was analyzed at concentrations of 5.0 and 10.0 mg/ml (0.78 and 1.56 mm ARNT-bHLH monomer, respectively) in its storage buffer (40 mm KCl, 2 mm dithiothreitol, 0.4 mm EDTA, 5% glycerol, and 20 mm Tris, pH 7.4). Peptide-DNA experiments were performed by mixing 1.56 mmARNT-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). Reported scattering values are the averages of at least 25 scans of 30 s each. All data were analyzed using AutoPro 4.0 PC software (Protein Solutions, Inc.). Dynamic light scattering uses the Brownian motion of molecules in solution, which causes scattered light intensity to fluctuate (35Schurr J.M. CRC Crit. Rev. Biochem. 1977; 4: 371-431Crossref PubMed Scopus (101) Google Scholar). These fluctuations are measured by the DynaPro instrument at 20 different times between 3 and 3000 µs. An exponential decay function is generated from the scattering data. The rate of decay of this function is used to determine the translational diffusion coefficient,DT. The radius of hydration, RH, is then calculated using the following DT =kT/6πηRH, where k is the Boltzmann constant, T is temperature in Kelvin, and η is the solvent viscosity. RH is defined as the radius of a hypothetical hard sphere that diffuses with the same speed as the particle under examination. However, macromolecules are non-spherical and solvated. Therefore, the molecular weight (Mr) of a macromolecule is estimated usingMrversus RHcalibration curves developed from standards of known molecular weight and size. Thus, the Mr estimate of a given particle is subject to error if it deviates from the shape and solvation of the molecules used as standards. The molecular weight for protein macromolecules is estimated from a curve that fits the equation Mr = [1.6800 ·RH]2.3398, as implemented in the AutoPro software. A similar calibration curve for oligonucleotides has not yet been developed and, hence, precludes an analysis of the ARNDNA alone. Sedimentation equilibrium measurements were done with a Model E analytical ultracentrifuge (Beckman Corp.) at 25 °C. A solution of 5 µm ARNDNA and 10 µm ARNT-bHLH was diluted 1:1 and 1:2 in Buffer B (150 mm NaCl, 100 mmTris, pH 7.4). Buffer B was placed in the reference compartment, and sample solutions were loaded into sample compartments of double sector cells. The initial experiment was run at 20,000 rpm for 24 h because the heaviest species expected was a 38-kDa complex of an ARNT-bHLH tetramer bound to double-stranded DNA. The distribution of the components at equilibrium was determined by measuring the UV absorption of each cell at 280 nm. However, at this wavelength we were unable to differentiate between peptide or DNA alone and peptide-DNA complexes. Given the inability to distinguish DNA from poly(d(I·C]) peptide at 280 nm, we used 5′-fluorescein-labeled DNA in some studies. Samples with 10 µm fluoresceinated ARNDNA and 40 µm ARNT-bHLH peptide were allowed to equilibrate for 20 h at 20,000 rpm. The samples were monitored at 494 nm, the absorption maximum of fluorescein. Only fluoresceinated DNA and peptide-DNA complexes are observed at this wavelength, without interference from free peptide. Data acquisition and analyses were done with Ultrascan software (Borries Demeler), and Scientist (MicroMath) was used for experimental data fitting. Weight average molecular weight for a single species model was calculated using the formula. C=C0+(CM−C0)·eMA(1−νρ)ω2(r2−rM2)/2RTEquation 4 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, ris the distance between each point along the concentration gradient and the rotor axis, and C0 and CMare the concentrations at points r andrM, respectively. ω, ν, ρ, (1 − νρ),R, and T represent angular velocity, partial specific volume, density, buoyancy, gas constant, and temperature, respectively. 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. 5Ferré-D'Amaré A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar, 6Ferré-D'Amaré A.R. Pognonec P. Roeder R.G. Burley S.K. EMBO J. 1994; 13: 180-189Crossref PubMed Scopus (333) Google Scholar, 7Ma P.C.M. Rould M.A. Weintraub H. Pabo C.O. Cell. 1994; 77: 451-459Abstract Full Text PDF PubMed Scopus (401) Google Scholar, 8Shimizu T. Toumoto A. Ihara K. Shimizu M. Kyogoku Y. Ogawa N. Oshima Y. Hakoshima T. EMBO J. 1997; 16: 4689-4697Crossref PubMed Scopus (155) Google Scholar, 9Ellenberger T.E. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Crossref PubMed Scopus (357) Google Scholar, 10Párraga A. Bellsolell L. Ferré-D'Amaré A.R. Burley S.K. Structure (Lond.). 1998; 6: 661-672Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Fig.1). CD spectra were measured to determine the secondary structure content of the ARNT-bHLH peptide and to observe any changes in secondary structure upon binding to DNA. CD spectra were also taken in the presence of TFE. TFE increases helicity of polypeptides by selectively destabilizing solvent-amide group interactions. Compact conformations such as α helices, which maximize intramolecular polypeptide backbone hydrogen bonding and lessen solvent exposure are favored (36Cammers-Goodwin A. Allen J.T. Oslick S.L. McClure K.F. Lee J.H. Kemp D.S. J. Am. Chem. Soc. 1996; 118: 3082-3090Crossref Scopus (242) Google Scholar). Medium-sized peptides with an intrinsic tendency to assume a helical conformation in water show an increase in helicity upon the addition of TFE. Hence, the CD spectra of the peptide with and without 50% TFE were measured to affirm that the synthetic peptide had the ability to adopt a helical structure. In both cases, spectra were obtained with strong maxima at 190 nm and double minima at 200–210 and 222 nm, characteristic of α helices. The amplitude of the spectrum for ARNT-bHLH with TFE was approximately three times that of peptide in buffer alone (Fig.2A). Secondary structure analysis using the variable selection method showed that the helicity increased from 12.8 to 68.0% upon the addition of TFE (TableI). The predicted maximum helicity attainable by the peptide is 82.1%. This maximum is calculated by assuming that the basic region, helix 1, and helix 2 (Fig. 1) would be completely α-helical, as observed in the crystal structu" @default.
• W2003075282 created "2016-06-24" @default.
• W2003075282 creator A5045847541 @default.
• W2003075282 creator A5061848565 @default.
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• W2003075282 date "2001-11-01" @default.
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• W2003075282 title "The Basic Helix-Loop-Helix Domain of the Aryl Hydrocarbon Receptor Nuclear Transporter (ARNT) Can Oligomerize and Bind E-box DNA Specifically" @default.
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• W2003075282 doi "https://doi.org/10.1074/jbc.m105675200" @default.
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