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In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor

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Please use this identifier to cite or link to this item:http://hdl.handle.net/2115/61643

Title: In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor
Authors: Tsutsumi, Motosuke Browse this author
Muto, Hideki Browse this author
Myoba, Shohei Browse this author
Kimoto, Mai Browse this author
Kitamura, Akira Browse this author
Kamiya, Masakatsu Browse this author
Kikukawa, Takashi Browse this author
Takiya, Shigeharu Browse this author
Demura, Makoto Browse this author
Kawano, Keiichi Browse this author
Kinjo, Masataka Browse this author
Aizawa, Tomoyasu Browse this author →KAKEN DB
Keywords: DNA-binding protein
fluorescence correlation spectroscopy
one score and three peptide repeat domain
silkworm
transcription factor
Issue Date: Feb-2016
Publisher: Wiley-Blackwell
Journal Title: Febs open bio
Volume: 6
Issue: 2
Start Page: 106
End Page: 125
Publisher DOI: 10.1002/2211-5463.12026
Abstract: Fibroin modulator-binding protein 1 (FMBP-1) is a silkworm transcription factor that has a unique DNA-binding domain called the one score and three amino acid peptide repeat (STPR). Here we used fluorescence correlation spectroscopy (FCS) to analyze the diffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein (EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at the same developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA-bound component and three discernible diffusion components. We also used FCS to observe the movements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP-1 mutants (e.g. mutants with N-terminal or C-terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP-1 states: (a) diffusion of free protein, (b) and (c) two types of transient interactions between FMBP-1 and chromosomal DNA, and (d) stable binding of FMBP-1 to chromosomal DNA.
Rights: https://creativecommons.org/licenses/by/4.0/
Type: article
URI: http://hdl.handle.net/2115/61643
Appears in Collections:生命科学院・先端生命科学研究院 (Graduate School of Life Science / Faculty of Advanced Life Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 相沢 智康

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