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Int. J. Mol. Sci. 2015, 16(7), 15743-15760; doi:10.3390/ijms160715743

A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR

1
Division of Structural Biology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan
2
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
3
Division of Structural Biology, Graduate School of Medicine, Kobe University, Kusunoki-cho, 7-5-1, Chuo-ku, Kobe 650-0017, Japan
4
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
5
Pharmaceutical Education Research Center, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
6
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan
7
Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
8
Graduate School of Information Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editors: Lukasz Kurgan and Vladimir N. Uversky
Received: 30 March 2015 / Revised: 17 June 2015 / Accepted: 1 July 2015 / Published: 10 July 2015
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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. View Full-Text
Keywords: solution nuclear magnetic resonance (NMR); intrinsically disordered proteins; protein flexibility; membrane protein; rotational correlation time solution nuclear magnetic resonance (NMR); intrinsically disordered proteins; protein flexibility; membrane protein; rotational correlation time
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Goda, N.; Shimizu, K.; Kuwahara, Y.; Tenno, T.; Noguchi, T.; Ikegami, T.; Ota, M.; Hiroaki, H. A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR. Int. J. Mol. Sci. 2015, 16, 15743-15760.

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