title: Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells.
abstract: Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.
authors.affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku 980-8578, Sendai, Japan.
authors.forename: Shuya
authors.initials: S
authors.lastname: Kasai
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku 980-8578, Sendai, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Kajimoto
authors.affiliation: School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku 226-8501, Yokohama, Japan.
authors.forename: Yuma
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku 980-8578, Sendai, Japan.
authors.forename: Tomo
authors.initials: T
authors.lastname: Saito
authors.affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku 980-8578, Sendai, Japan.
authors.forename: Ken-Ichi
authors.initials: KI
authors.lastname: Yasumoto
authors.affiliation: School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku 226-8501, Yokohama, Japan.
authors.forename: Makio
authors.initials: M
authors.lastname: Tokunaga
authors.affiliation: School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku 226-8501, Yokohama, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Sakata-Sogawa
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku 980-8578, Sendai, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fukumura
authors.affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku 980-8578, Sendai, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Sogawa
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D003412:Cricetulus; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins; D051020:bcl-X Protein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012097:Repressor Proteins; D051020:bcl-X Protein; C494811:BCL2L1 protein, human; C497442:HIF1A protein, human; C116973:HIF3A protein, human
keywords: FLIM-FRET analysis; conformational change; dual-color single molecule imaging