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Multiple replacements at amino acid position 3 of bacteriophage T4 lysozyme have
shown that the conformational stability of the protein is directly governed by
the hydrophobicity of the residue substituted (Matsumura, M., Becktel, W. J.,
and Matthews, B. W. (1988) Nature 334, 406-410). Of the 13 mutant lysozymes made
by site-directed mutagenesis, two variants, one with valine (I3V) and the other
with tyrosine (I3Y), were crystallized and their structures solved. In this
report we describe the crystal structures of these variants at 1.7 A resolution.
While the structure of the I3V mutant is essentially the same as that of
wild-type lysozyme, the I3Y mutant has substantial changes in its structure. The
most significant of these are that the side chain of the tyrosine is not
accommodated within the interior of the protein and the amino-terminal
polypeptide (residues 1-9) moves 0.6-1.1 A relative to the wild-type structure.
Using coordinates based on the wild-type and available mutant structures,
solvent accessible surface area of residue 3 as well as the adjacent 9 residues
in the folded form were calculated. The free energy of stabilization based on
the transfer of these residues from a fully extended form to the interior to the
folded protein was found to correlate well with the protein stability determined
by thermodynamic analysis. The enhanced thermostability of the variant
Ile-3----Leu, relative to wild-type lysozyme, can also be rationalized by
surface-area calculations based on a model-built structure. Noncrystallization
of most lysozyme variants at position 3 appears to be due to disruption of
intermolecular contacts in the crystal. The Ile-3----Val variant is closely
isomorphous with wild-type and maintains the same crystal contacts. In the
Ile-3----Tyr variant, however, a new set of contacts is made in which direct
protein-protein hydrogen bonds are replaced by protein-water-protein hydrogen
bonds as well as a novel hydrogen bond involving the phenolic hydroxyl of the
substituted tyrosine.
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