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Journal of Biomolecular NMR

, Volume 37, Issue 1, pp 53–63 | Cite as

Fine-tuning of protein domain boundary by minimizing potential coiled coil regions

  • Naoko Iwaya
  • Natsuko Goda
  • Satoru Unzai
  • Kenichiro Fujiwara
  • Toshiki Tanaka
  • Kentaro Tomii
  • Hidehito Tochio
  • Masahiro Shirakawa
  • Hidekazu HiroakiEmail author
Original Paper

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.

Keywords

Domain boundary determination Hydrophobic interaction HSQC Nonspecific self-association 

Abbreviations

8-ANS

8-Anilino-1-naphthalenesulfonic acid

HSQC

Heteronuclear single quantum correlation spectroscopy

PCR

Polymerase chain reaction

AAA

ATPase associated with various cellular activities

KP60

katanin p60

NVL2

Nuclear VCP-like protein 2

VCP

Valosin containing protein p97

GST

Glutathione S-transferase

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Notes

Acknowledgments

This work was partly supported by grants to H.H. from the Japanese Ministry of Education, Science, Sports and Culture (Protein3000), and was supported by grants to H.H. and K.T. from Japan Science and Technology Agency (BIRD). We thank Mr. K. Inomata for help with data representation.

Supplementary material

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Naoko Iwaya
    • 1
    • 2
  • Natsuko Goda
    • 1
  • Satoru Unzai
    • 1
  • Kenichiro Fujiwara
    • 1
  • Toshiki Tanaka
    • 3
  • Kentaro Tomii
    • 4
  • Hidehito Tochio
    • 1
    • 2
  • Masahiro Shirakawa
    • 1
    • 2
  • Hidekazu Hiroaki
    • 1
    Email author
  1. 1.Field of Supramolecular Biology, International Graduate School of Arts and SciencesYokohama City UniversityYokohama, KanagawaJapan
  2. 2.Department of Molecular Engineering, Graduate School of EngineeringKyoto UniversityKatsura, KyotoJapan
  3. 3.Graduate School of Material ScienceOMOHI-college, Nagoya Institute of TechnologyGokiso-cho, NagoyaJapan
  4. 4.Computational Biology Research CenterThe National Institute of Advanced Industrial Science and TechnologyAomi, Koto-ku, TokyoJapan

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