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-alpha-domain disulfide bridges, C6-C127 and C30-C115, is partially folded, with the alpha domain tightly folded to the nativelike conformation and the beta 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 beta sheet and the 3(10) helix in the beta 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 alpha domain was prevented by the C helix from propagating toward the beta domain. A marginally stable state in folded proteins is discussed based on the structures remaining in each three-disulfide variant.
Yokota
Atsushi
A
Japan Science and Technology Corporation, Izumi, Osaka, 594-1144, Japan.
Hirai
Kenichi
K
Miyauchi
Hiroyo
H
Iimura
Satoshi
S
Noda
Yasuo
Y
Inoue
Kyoko
K
Akasaka
Kazuyuki
K
Tachibana
Hideki
H
Segawa
Shin-ichi
S
D000577:Amides / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding
D000577:Amides; D004220:Disulfides; D006859:Hydrogen; D009113:Muramidase