Fine-tuning of protein domain boundary by minimizing potential coiled coil regions
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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.
KeywordsDomain boundary determination Hydrophobic interaction HSQC Nonspecific self-association
Heteronuclear single quantum correlation spectroscopy
Polymerase chain reaction
ATPase associated with various cellular activities
Nuclear VCP-like protein 2
Valosin containing protein p97
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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.
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