title: A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR.
abstract: Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible have attracted the attention of biologists. Therefore, the development of a systematic method to identify polypeptide regions that are unstructured in solution is important. We have designed an "indirect/reflected" detection system for evaluating the physicochemical properties of IDPs using nuclear magnetic resonance (NMR). This approach employs a "chimeric membrane protein"-based method using the thermostable membrane protein PH0471. This protein contains two domains, a transmembrane helical region and a C-terminal OB (oligonucleotide/oligosaccharide binding)-fold domain (named NfeDC domain), connected by a flexible linker. NMR signals of the OB-fold domain of detergent-solubilized PH0471 are observed because of the flexibility of the linker region. In this study, the linker region was substituted with target IDPs. Fifty-three candidates were selected using the prediction tool POODLE and 35 expression vectors were constructed. Subsequently, we obtained 15N-labeled chimeric PH0471 proteins with 25 IDPs as linkers. The NMR spectra allowed us to classify IDPs into three categories: flexible, moderately flexible, and inflexible. The inflexible IDPs contain membrane-associating or aggregation-prone sequences. This is the first attempt to use an indirect/reflected NMR method to evaluate IDPs and can verify the predictions derived from our computational tools.
authors.affiliation: Division of Structural Biology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. tenno.natsuko@f.mbox.nagoya-u.ac.jp.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Goda
authors.affiliation: Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo Waterfront Bio-IT Research Building 2-4-7 Aomi, Koto-ku, Tokyo 135-0046, Japan. shimizu-kana@aist.go.jp.
authors.forename: Kana
authors.initials: K
authors.lastname: Shimizu
authors.affiliation: Division of Structural Biology, Graduate School of Medicine, Kobe University, Kusunoki-cho, 7-5-1, Chuo-ku, Kobe 650-0017, Japan. y_kuwahara11@hotmail.com.
authors.forename: Yohta
authors.initials: Y
authors.lastname: Kuwahara
authors.affiliation: The Structural Biology Research Center and Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. tenno.takeshi@e.mbox.nagoya-u.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tenno
authors.affiliation: Pharmaceutical Education Research Center, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. noguchit@my-pharm.ac.jp.
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Noguchi
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan. ikegamit@tsurumi.yokohama-cu.ac.jp.|Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. ikegamit@tsurumi.yokohama-cu.ac.jp.
authors.forename: Takahisa
authors.initials: T
authors.lastname: Ikegami
authors.affiliation: Graduate School of Information Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. mota@is.nagoya-u.ac.jp.
authors.forename: Motonori
authors.initials: M
authors.lastname: Ota
authors.affiliation: Division of Structural Biology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. hiroaki.hidekazu@f.mbox.nagoya-u.ac.jp.|Division of Structural Biology, Graduate School of Medicine, Kobe University, Kusunoki-cho, 7-5-1, Chuo-ku, Kobe 650-0017, Japan. hiroaki.hidekazu@f.mbox.nagoya-u.ac.jp.|The Structural Biology Research Center and Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. hiroaki.hidekazu@f.mbox.nagoya-u.ac.jp.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Hiroaki
mesh_terms: D000818:Animals; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008962:Models, Theoretical; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary
chemical_list: D064267:Intrinsically Disordered Proteins
keywords: intrinsically disordered proteins; membrane protein; protein flexibility; rotational correlation time; solution nuclear magnetic resonance (NMR)