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PDBsum entry 2zwo
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References listed in PDB file
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Key reference
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Title
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Requirement of a unique ca(2+)-Binding loop for folding of tk-Subtilisin from a hyperthermophilic archaeon.
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Authors
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Y.Takeuchi,
S.Tanaka,
H.Matsumura,
Y.Koga,
K.Takano,
S.Kanaya.
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Ref.
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Biochemistry, 2009,
48,
10637-10643.
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PubMed id
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Abstract
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Tk-subtilisin from the hyperthermophiolic archaeon Thermococcus kodakaraensis
matures from Pro-Tk-subtilisin upon autoprocessing and degradation of
Tk-propeptide [Tanaka, S., Saito, K., Chon, H., Matsumura, H., Koga, Y., Takano,
K., and Kanaya, S. (2007) J. Biol. Chem. 282, 8246-8255]. It requires Ca(2+) for
folding and assumes a molten globule-like structure in the absence of Ca(2+)
even in the presence of Tk-propeptide. Tk-subtilisin contains seven
Ca(2+)-binding sites. Four of them (Ca2-Ca5) are located within a long loop,
which mostly consists of a unique insertion sequence of this protein. To analyze
the role of this Ca(2+)-binding loop, three mutant proteins,
Deltaloop-Tk-subtilisin, DeltaCa2-Pro-S324A, and DeltaCa3-Pro-S324A, were
constructed. These proteins were designed to remove the Ca(2+)-binding loop, Ca2
site, or Ca3 site of Pro-Tk-subtilisin or its active site mutant Pro-S324A.
Far-UV CD spectra of these proteins refolded in the absence and presence of
Ca(2+) indicated that Deltaloop-Tk-subtilisin completely lost the ability to
fold into a native structure. In contrast, two other proteins retained this
ability, although their refolding rates were greatly decreased compared to that
of Pro-S324A. Determination of the crystal structures of these proteins purified
in a Ca(2+)-bound form indicates that the structures of DeltaCa2-Pro-S324A and
DeltaCa3-Pro-S324A are virtually identical to that of Pro-S324A, except that
they lack the Ca2 and Ca3 sites, respectively, and the structure of the
Ca(2+)-binding loop is destabilized. Nevertheless, these proteins were slightly
more stable than Pro-S324A. These results suggest that the Ca(2+)-binding loop
is required for folding of Tk-subtilisin but does not seriously contribute to
the stabilization of Tk-subtilisin in a native structure.
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