Characterisation of Disulfide‐Bond Dynamics in Non‐Native States of Lysozyme and Its Disulfide Deletion Mutants by NMR

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.

Characterisation of the Conformational Properties of Urea-unfolded Im7: Implications for the Early Stages of Protein Folding

“…Importantly, the properties of the remaining clusters of H3G3 Im7* are similar to those of the wild-type protein, as are the clusters of Phe15Ala Im7. Taken together, the data suggest that the clusters in urea-unfolded Im7 do not interact with each other to any significant extent, in marked contrast to the results obtained previously for ureaunfolded lysozyme, 11,50 and the colicin E9 translocation domain. 21 In both these cases, however, the major clusters that interact are centred on tryptophan residues, whereas in urea-unfolded Im7, which only has one tryptophan residue, the four clusters are formed around smaller, mostly aliphatic groups, which may reduce the solvent entropic factors that lead to collapse of the clusters in these proteins.…”

“…11,50 In order to determine if the clusters of urea-unfolded Im7 show similar behaviour, we studied two variants of Im7: H3G3 Im7*, in which residues 51-56 that form helix III in the native protein have been replaced by three glycine residues (this protein also contains an additional N-terminal His 6 tag), 51 and Phe15Ala Im7. These variants were selected because cluster III has been removed from H3G3 Im7*, while Phe15 is both the largest residue in cluster I of the wild-type protein and, should the individual clusters interact, would be most likely to show an effect on the chemical shifts (due to removal of the ring-current effect) or dynamic properties (due to its large hydrophobic surface area) of resonances from other clusters.…”

Conserved Folding Pathways of α-Lactalbumin and Lysozyme Revealed by Kinetic CD, Fluorescence, NMR, and Interrupted Refolding Experiments

“…[55][56][57][58] An essential role is played by the W62W63 motif located at the interface of the α-subdomain and the β-subdomain in the native structure of lysozyme, exerting a stabilizing effect on hydrophobic clusters in the α-domain via a long-range interaction in the unfolded state. 56,57 We have recently discussed the possible role of this interaction in guiding the protein toward the folded state by mediating intersubdomain interactions.…”

Mass spectrometry profiles superoxide-induced intramolecular disulfide in the FMN-binding subunit of mitochondrial complex I

“…X-ray crystallography and NMR are excellent tools for studying the 3D structure of proteins, thus providing information regarding disulfide linkages [30–33]. However, large amounts of well purified protein with good solubility or the proper size crystal are required for these two methods respectively [34].…”