NMR Characterization of Three-Disulfide Variants of Lysozyme, C64A/C80A, C76A/C94A, and C30A/C115AA Marginally Stable State in Folded Proteins

Our earlier NMR study showed that a two-disulfide variant of hen lysozyme containing intra-α-domain disulfide bridges, C6−C127 and C30−C115, is partially folded, with the α domain tightly folded to the nativelike conformation and the β domain flexible or unfolded. With a view that the formation of a third disulfide bridge is a key for the accomplishment of the overall chain fold, three-dimensional structures of three-disulfide variants of hen lysozyme lacking one disulfide bridge (C64A/C80A, C76A/C94A, and C30A/C115A) were studied in detail using NMR spectroscopy. Amide hydrogen exchange rates were measured to estimate the degree of conformational fluctuation in a residue-specific manner. The structure of C76A/C94A was found to be quite similar to that of the wild type, except for the peptide segment of residues 74−78. The structure of C64A/C80A was considerably disordered in the entire region of the loop (residues 62−79). Further, it was found that a network of hydrogen bonds within the β sheet and the 3₁₀ helix in the β domain were disrupted and fluctuating. In C30A/C115A, the D helix was unstructured and the interface of the B helix with the D helix was significantly perturbed. However, the structural disorder generated in the hydrophobic core of the α domain was prevented by the C helix from propagating toward the β domain. A marginally stable state in folded proteins is discussed based on the structures remaining in each three-disulfide variant.