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A disulfide bond introduced between amino acid positions 9 and 164 in phage T4
lysozyme has been shown to significantly increase the stability of the enzyme
toward thermal denaturation [Matsumura, M., Becktel, W.J., Levitt, M., &
Matthews, B. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6562-6566]. To
elucidate the structural features of the engineered disulfide, the crystal
structure of the disulfide mutant has been determined at 1.8-A resolution.
Residue 9 lies in the N-terminal alpha-helix, while residue 164 is located at
the extreme C terminus of T4 lysozyme, which is the most mobile part of the
molecule. The refined structure shows that the formation of the disulfide bond
is accompanied by relatively large (approximately 2.5 A) localized shifts in
C-terminal main-chain atoms. Comparison of the geometry of the engineered
disulfide with those of naturally observed disulfides in proteins shows that the
engineered bridge adopts a left-handed spiral conformation with a typical set of
dihedral angles and C alpha-C alpha distance. The geometry of the engineered
disulfide suggests that it is slightly more strained than the disulfide of
oxidized dithiothreitol but that the strain is within the range observed in
naturally occurring disulfides. The wild-type and cross-linked lysozymes have
very similar overall crystallographic temperature factors, indicating that the
introduction of the disulfide bond does not impose rigidity on the folded
protein structure. In particular, residues 162-164 retain high mobility in the
mutant structure, consistent with the idea that stabilization of the protein is
due to the effect of the disulfide cross-link on the unfolded rather than the
folded state.(ABSTRACT TRUNCATED AT 250 WORDS)
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