title: Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix-loop-helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms.
abstract: Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated transcription factor with two heme-binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme-binding kinetics and DNA-binding characteristics of the isolated fragment containing the N-terminal basic helix-loop-helix (bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the PAS-A domain alone. We found that the heme-bound bHLH-PAS-A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH-PAS-A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS-A (412 nm). The axial ligand trans to CO in bHLH-PAS-A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo-bHLH-PAS (3.3 x 10(7) mol(-1) x s(-1)) was more than two orders of magnitude higher than for association with apo-PAS-A (< 10(5) mol(-1) x s(-1)). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)-NO heme complex is five-coordinated. Using the quartz-crystal microbalance method, we found that the bHLH-PAS-A domain binds specifically to the E-box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH-PAS-A and its potential role in regulating DNA binding.
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Mukaiyama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchida
authors.forename: Emiko
authors.initials: E
authors.lastname: Sato
authors.forename: Ai
authors.initials: A
authors.lastname: Sasaki
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.forename: Jotaro
authors.initials: J
authors.lastname: Igarashi
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kurokawa
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sagami
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002940:Circadian Rhythm / Q000502:physiology*; D004247:DNA / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000502:physiology*; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D006418:Heme; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C491259:Npas2 protein, mouse