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Title
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Structural studies of mutants of the lysozyme of bacteriophage T4. The temperature-sensitive mutant protein Thr157----Ile.
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Authors
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M.G.Grütter,
T.M.Gray,
L.H.Weaver,
T.A.Wilson,
B.W.Matthews.
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Ref.
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J Mol Biol, 1987,
197,
315-329.
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PubMed id
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Abstract
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To understand the roles of individual amino acids in the folding and stability
of globular proteins, a systematic structural analysis of mutants of the
lysozyme of bacteriophage T4 has been undertaken. The isolation,
characterization, crystallographic refinement and structural analysis of a
temperature-sensitive lysozyme in which threonine 157 is replaced by isoleucine
is reported here. This mutation reduces the temperature of the midpoint of the
reversible thermal denaturation transition by 11 deg.C at pH 2.0. Electron
density maps showing differences between the wild-type and mutant X-ray crystal
structures have obvious features corresponding to the substitution of threonine
157 by isoleucine. There is little difference electron density in the remainder
of the molecule, indicating that the structural changes are localized to the
site of the mutation. High-resolution crystallographic refinement of the mutant
lysozyme structure confirms that it is very similar to wild-type lysozyme. The
largest conformational differences are in the gamma-carbon of residue 157 and in
the side-chain of Asp159, which shift 1.0 A and 1.1 A, respectively. In the
wild-type enzyme, the gamma-hydroxyl group of Thr157 participates in a network
of hydrogen bonds. Substitution of Thr157 with an isoleucine disrupts this set
of hydrogen bonds. A water molecule bound in the vicinity of Thr155 partially
restores the hydrogen bond network in the mutant structure, but the buried
main-chain amide of Asp159 is not near a hydrogen bond acceptor. This
unsatisfied hydrogen-bonding potential is the most obvious reason for the
reduction in stability of the temperature-sensitive mutant protein.
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