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Title
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Tolerance of T4 lysozyme to proline substitutions within the long interdomain alpha-helix illustrates the adaptability of proteins to potentially destabilizing lesions.
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Authors
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U.H.Sauer,
D.P.San,
B.W.Matthews.
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Ref.
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J Biol Chem, 1992,
267,
2393-2399.
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PubMed id
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Abstract
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To investigate the ability of a protein to accommodate potentially destabilizing
amino acid substitutions, and also to investigate the steric requirements for
catalysis, proline was substituted at different sites within the long
alpha-helix that connects the amino-terminal and carboxyl-terminal domains of T4
lysozyme. Of the four substitutions attempted, three yielded folded, functional
proteins. The catalytic activities of these three mutant proteins (Q69P, D72P,
and A74P) were 60-90% that of wild-type. Their melting temperatures were 7-12
degrees C less than that of wild-type at pH 6.5. Mutant D72P formed crystals
isomorphous with wild-type allowing the structure to be determined at high
resolution. In the crystal structure of wild-type lysozyme the interdomain
alpha-helix has an overall bend angle of 8.5 degrees. In the mutant structure
the introduction of the proline causes this bend angle to increase to 14 degrees
and also causes a corresponding rotation of 5.5 degrees of carboxyl-terminal
domain relative to the amino-terminal one. Except for the immediate location of
the proline substitution there is very little change in the geometry of the
interdomain alpha-helix. The results support the view that protein structures
are adaptable and can compensate for potentially destabilizing amino acid
substitutions. The results also suggest that the precise shape of the active
site cleft of T4 lysozyme is not critical for catalysis.
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