TY - JOUR
T1 - A method for systematic assessment of intrinsically disordered protein regions by NMR
AU - Goda, Natsuko
AU - Shimizu, Kana
AU - Kuwahara, Yohta
AU - Tenno, Takeshi
AU - Noguchi, Tamotsu
AU - Ikegami, Takahisa
AU - Ota, Motonori
AU - Hiroaki, Hidekazu
N1 - Publisher Copyright:
© 2015 by the authors; licensee MDPI, Basel, Switzerland.
PY - 2015/7/10
Y1 - 2015/7/10
N2 - 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.
AB - 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.
KW - Intrinsically disordered proteins
KW - Membrane protein
KW - Protein flexibility
KW - Rotational correlation time
KW - Solution nuclear magnetic resonance (NMR)
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U2 - 10.3390/ijms160715743
DO - 10.3390/ijms160715743
M3 - Article
C2 - 26184172
AN - SCOPUS:84939506644
VL - 16
SP - 15743
EP - 15760
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
SN - 1661-6596
IS - 7
ER -