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PDBsum entry 1a2q
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References listed in PDB file
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Key reference
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Title
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Large increases in general stability for subtilisin bpn' Through incremental changes in the free energy of unfolding.
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Authors
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M.W.Pantoliano,
M.Whitlow,
J.F.Wood,
S.W.Dodd,
K.D.Hardman,
M.L.Rollence,
P.N.Bryan.
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Ref.
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Biochemistry, 1989,
28,
7205-7213.
[DOI no: ]
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PubMed id
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Abstract
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Six individual amino acid substitutions at separate positions in the tertiary
structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability
of this enzyme, as judged by differential scanning calorimetry and decreased
rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K,
M50F, Q206C, and N76D, were discovered through the use of five different
investigative approaches: (1) random mutagenesis; (2) design of buried
hydrophobic side groups; (3) design of electrostatic interactions at Ca2+
binding sites; (4) sequence homology consensus; and (5) serendipity.
Individually, the six amino acid substitutions increase the delta G of unfolding
between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six
individual stabilizing mutations together into one subtilisin BPN' molecule was
found to result in approximately independent and additive increases in the delta
G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C).
Thermodynamic stability was also shown to be related to resistance to
irreversible inactivation, which included elevated temperatures (65 degrees C)
or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of
inactivation of the combination variant is approximately 300 times slower than
that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution
crystal structures of mutant and wild-type enzymes revealed only independent and
localized structural changes around the site of the amino acid side group
substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)
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Secondary reference #1
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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.
[DOI no: ]
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PubMed id
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Secondary reference #2
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Title
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Proteases of enhanced stability: characterization of a thermostable variant of subtilisin.
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Authors
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P.N.Bryan,
M.L.Rollence,
M.W.Pantoliano,
J.Wood,
B.C.Finzel,
G.L.Gilliland,
A.J.Howard,
T.L.Poulos.
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Ref.
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Proteins, 1986,
1,
326-334.
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PubMed id
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Secondary reference #3
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Title
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Atomic coordinates for subtilisin bpn' (Or novo).
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Authors
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R.A.Alden,
J.J.Birktoft,
J.Kraut,
J.D.Robertus,
C.S.Wright.
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Ref.
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Biochem Biophys Res Commun, 1971,
45,
337-344.
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PubMed id
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