tailieunhanh - Báo cáo khoa học: 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

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. | iFEBS Journal 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 Yuji Mukaiyama1 Takeshi Uchida2 Emiko Sato1 Ai Sasaki1 Yuko Sato1 Jotaro Igarashi1 Hirofumi Kurokawa1 Ikuko Sagami1t Teizo Kitagawa2 and Toru Shimizu1 1 Institute of Multidisciplinary Research for Advanced Materials Tohoku University Sendai Japan 2 Okazaki Institute for Integrative Bioscience NationalInstitutes of NaturalSciences Okazaki Japan Keywords circadian rhythms DNA binding hemesensor protein PAS domain resonance Raman spectroscopy Correspondence T. Shimizu Institute of Multidisciplinary Research for Advanced Materials Tohoku University 2-1-1 Katahira Aoba-ku Sendai 980-8577 Japan Fax 81 22 217 5604 5390 Tel 81 22 217 5604 5605 E-mail shimizu@ Present address Division of Chemistry Graduate Schoolof Science Hokkaido University Kita-ku Sapporo Japan Graduate Schoolof Agriculture Kyoto PrefecturalUniversity Shimogamo Sakyo-ku Kyoto Japan Received 23 February 2006 accepted 4 April 2006 doi 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 .