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

被引:6
|
作者
Goda, Natsuko [1 ]
Shimizu, Kana [2 ]
Kuwahara, Yohta [3 ]
Tenno, Takeshi [4 ,5 ]
Noguchi, Tamotsu [6 ]
Ikegami, Takahisa [7 ,8 ]
Ota, Motonori [9 ]
Hiroaki, Hidekazu [1 ,3 ,4 ,5 ]
机构
[1] Nagoya Univ, Grad Sch Pharmaceut Sci, Div Struct Biol, Chikusa Ku, Nagoya, Aichi 4648601, Japan
[2] Natl Inst Adv Ind Sci & Technol, Computat Biol Res Ctr, Koto Ku, Tokyo 1350046, Japan
[3] Kobe Univ, Grad Sch Med, Div Struct Biol, Chuo Ku, Kobe, Hyogo 6500017, Japan
[4] Nagoya Univ, Struct Biol Res Ctr, Grad Sch Sci, Chikusa Ku, Nagoya, Aichi 4648601, Japan
[5] Nagoya Univ, Div Biol Sci, Grad Sch Sci, Chikusa Ku, Nagoya, Aichi 4648601, Japan
[6] Meiji Pharmaceut Univ, Pharmaceut Educ Res Ctr, Tokyo 2048588, Japan
[7] Osaka Univ, Inst Prot Res, Suita, Osaka 5650871, Japan
[8] Yokohama City Univ, Grad Sch Med Life Sci, Tsurumi Ku, Yokohama, Kanagawa 2300045, Japan
[9] Nagoya Univ, Grad Sch Informat Sci, Chikusa Ku, Nagoya, Aichi 4648601, Japan
来源
基金
日本学术振兴会;
关键词
solution nuclear magnetic resonance (NMR); intrinsically disordered proteins; protein flexibility; membrane protein; rotational correlation time; UNSTRUCTURED PROTEINS; PREDICTION; DOMAINS; SPECTROSCOPY; DATABASE; BINDING; VECTOR; CLASSIFICATION; PURIFICATION; ENDOCYTOSIS;
D O I
10.3390/ijms160715743
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 N-15-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.
引用
收藏
页码:15743 / 15760
页数:18
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