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To clarify the role of amino acid residues at turns in the conformational
stability and folding of a globular protein, six mutant human lysozymes deleted
or substituted at turn structures were investigated by calorimetry, GuHCl
denaturation experiments, and X-ray crystal analysis. The thermodynamic
properties of the mutant and wild-type human lysozymes were compared and
discussed on the basis of their three-dimensional structures. For the deletion
mutants, Delta47-48 and Delta101, the deleted residues are in turns on the
surface and are absent in human alpha-lactalbumin, which is homologous to human
lysozyme in amino acid sequence and tertiary structure. The stability of both
mutants would be expected to increase due to a decrease in conformational
entropy in the denatured state; however, both proteins were destabilized. The
destabilizations were mainly caused by the disappearance of intramolecular
hydrogen bonds. Each part deleted was recovered by the turn region like the
alpha-lactalbumin structure, but there were differences in the main-chain
conformation of the turn between each deletion mutant and alpha-lactalbumin even
if the loop length was the same. For the point mutants, R50G, Q58G, H78G, and
G37Q, the main-chain conformations of these substitution residues located in
turns adopt a left-handed helical region in the wild-type structure. It is
thought that the left-handed non-Gly residue has unfavorable conformational
energy compared to the left-handed Gly residue. Q58G was stabilized, but the
others had little effect on the stability. The structural analysis revealed that
the turns could rearrange the main-chain conformation to accommodate the
left-handed non-Gly residues. The present results indicate that turn structures
are able to change their main-chain conformations, depending upon the side-chain
features of amino acid residues on the turns. Furthermore, stopped-flow GuHCl
denaturation experiments on the six mutants were performed. The effects of
mutations on unfolding-refolding kinetics were significantly different among the
mutant proteins. The deletion/substitutions in turns located in the alpha-domain
of human lysozyme affected the refolding rate, indicating the contribution of
turn structures to the folding of a globular protein.
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