Fine-tuning of protein domain boundary by minimizing potential coiled coil regions |
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Authors: | Naoko Iwaya Natsuko Goda Satoru Unzai Kenichiro Fujiwara Toshiki Tanaka Kentaro Tomii Hidehito Tochio Masahiro Shirakawa Hidekazu Hiroaki |
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Affiliation: | (1) Field of Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29, Tsurumi, Yokohama, Kanagawa 230-0045, Japan;(2) Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 606-8501, Japan;(3) Graduate School of Material Science, OMOHI-college, Nagoya Institute of Technology, Gokiso-cho, Nagoya 466-8555, Japan;(4) Computational Biology Research Center, The National Institute of Advanced Industrial Science and Technology, Aomi, Koto-ku, Tokyo 135-0064, Japan |
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Abstract: | Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in 1H–15N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer–dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by 1H–15N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . |
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Keywords: | Domain boundary determination Hydrophobic interaction HSQC Nonspecific self-association |
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