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Title
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Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond.
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Authors
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M.W.Pantoliano,
R.C.Ladner,
P.N.Bryan,
M.L.Rollence,
J.F.Wood,
T.L.Poulos.
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Ref.
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Biochemistry, 1987,
26,
2077-2082.
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PubMed id
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Abstract
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Introduction of a disulfide bond by site-directed mutagenesis was found to
enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of
conditions. The location of the new disulfide bond was selected with the aid of
a computer program, which scored various sites according to the amount of
distortion that an introduced disulfide linkage would create in a 1.3-A X-ray
model of native subtilisin BPN'. Of the several amino acid pairs identified by
this program as suitable candidates, Thr-22 and Ser-87 were selected by using
the additional requirement that the individual cysteine substitutions occur at
positions that exhibit some degree of variability in related subtilisin amino
acid sequences. A subtilisin variant containing cysteine residues at positions
22 and 87 was created by site-directed mutagenesis and was shown to have an
activity essentially equivalent to that of the wild-type enzyme. Differential
scanning calorimetry experiments demonstrated the variant protein to have a
melting temperature 3.1 degrees C higher than that of the wild-type protein and
5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant
protein. Kinetic experiments performed under a variety of conditions, including
8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal
inactivation at half the rate of that of the wild-type enzyme. The increased
thermal stability of this disulfide variant is consistent with a decrease in
entropy for the unfolded state relative to the unfolded state that contains no
cross-link, as would be predicted from the statistical thermodynamics of
polymers.
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