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- Title
A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR.
- Authors
Natsuko Goda; Kana Shimizu; Yohta Kuwahara; Takeshi Tenno; Tamotsu Noguchi; Takahisa Ikegami; Motonori Ota; Hidekazu Hiroaki
- 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.
- Subjects
PROTEIN conformation; MEMBRANE proteins; HEAT stability in proteins; BIOLOGISTS; NUCLEAR magnetic resonance spectroscopy; C-terminal binding proteins
- Publication
International Journal of Molecular Sciences, 2015, Vol 16, Issue 7, p15743
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms160715743