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Title
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Evaluation at atomic resolution of the role of strain in destabilizing the temperature-sensitive T4 lysozyme mutant Arg 96 --> His.
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Authors
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B.H.Mooers,
D.E.Tronrud,
B.W.Matthews.
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Ref.
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Protein Sci, 2009,
18,
863-870.
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PubMed id
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Abstract
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Mutant R96H is a classic temperature-sensitive mutant of bacteriophage T4
lysozyme. It was in fact the first variant of the protein to be characterized
structurally. Subsequently, it has been studied extensively by a variety of
experimental and computational techniques, but the reasons for the loss of
stability of the mutant protein remain controversial. In the crystallographic
refinement of the mutant structure at 1.9 A resolution one of the bond angles at
the site of substitution appeared to be distorted by about 11( degrees ), and it
was suggested that this steric strain was one of the major factors in
destabilizing the mutant. Different computationally-derived models of the mutant
structure, however, did not show such distortion. To determine the geometry at
the site of mutation more reliably, we have extended the resolution of the data
and refined the wildtype (WT) and mutant structures to be better than 1.1 A
resolution. The high-resolution refinement of the structure of R96H does not
support the bond angle distortion seen in the 1.9 A structure determination. At
the same time, it does confirm other manifestations of strain seen previously
including an unusual rotameric state for His96 with distorted hydrogen bonding.
The rotamer strain has been estimated as about 0.8 kcal/mol, which is about 25%
of the overall reduction in stability of the mutant. Because of concern that
contacts from a neighboring molecule in the crystal might influence the geometry
at the site of mutation we also constructed and analyzed supplemental mutant
structures in which this crystal contact was eliminated. High-resolution
refinement shows that the crystal contacts have essentially no effect on the
conformation of Arg96 in WT or on His96 in the R96H mutant.
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