Publications (3)7.37 Total impact
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Article: Characterisation of disulfide-bond dynamics in non-native states of lysozyme and its disulfide deletion mutants by NMR.
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ABSTRACT: This report describes NMR-spectroscopic investigations of the conformational dynamics of disulfide bonds in hen-egg-white lysozyme substitution mutants. The following four systems have been investigated: 2SS(alpha), a lysozyme variant that contains C64A, C76A, C80A and C94A substitutions, was studied in water at pH 2 and 3.8 and in urea (8 M, pH 2); 2SS(beta) lysozyme, which has C6S, C30A, C115A and C127A substitutions, was studied in water (pH 2) and urea (8 M, pH 2). The NMR analysis of heteronuclear 15N-relaxation rates shows that the barrier to disulfide-bond isomerisation can vary substantially in different lysozyme mutants and depends on the residual structure present in these states. The investigations reveal cooperativity in the modulation of micro- to millisecond dynamics that is due to the presence of multiple disulfide bridges in lysozyme. Mutation of cysteines in one of the two structural domains substantially diminishes the barrier to rotational isomerisation in the other domain. However, the interactions between hydrophobic clusters within and across the domains remains intact.ChemBioChem 10/2005; 6(9):1619-27. · 3.94 Impact Factor -
Article: NMR characterization of three-disulfide variants of lysozyme, C64A/C80A, C76A/C94A, and C30A/C115A--a marginally stable state in folded proteins.
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ABSTRACT: 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.Biochemistry 07/2004; 43(21):6663-9. · 3.42 Impact Factor -
Article: NMR Characterization of Three-Disulfide Variants of Lysozyme, C64A/C80A, C76A/C94A, and C30A/C115AA Marginally Stable State in Folded Proteins
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ABSTRACT: 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.05/2004;
