A method for systematic assessment of intrinsically disordered protein regions by NMR

Natsuko Goda, Kana Shimizu, Yohta Kuwahara, Takeshi Tenno, Tamotsu Noguchi, Takahisa Ikegami, Motonori Ota, Hidekazu Hiroaki

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)15743-15760
Number of pages18
JournalInternational Journal of Molecular Sciences
Volume16
Issue number7
DOIs
Publication statusPublished - 2015 Jul 10
Externally publishedYes

Keywords

  • Intrinsically disordered proteins
  • Membrane protein
  • Protein flexibility
  • Rotational correlation time
  • Solution nuclear magnetic resonance (NMR)

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Spectroscopy
  • Inorganic Chemistry
  • Catalysis
  • Molecular Biology
  • Computer Science Applications

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