Characterisation of disulfide-bond dynamics in non-native states of lysozyme and its disulfide deletion mutants by NMR.
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.
Collins
Emily S
ES
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe University, Marie-Curie-Strasse 11, 60439 Frankfurt am Main, Germany.
Wirmer
Julia
J
Hirai
Kenichi
K
Tachibana
Hideki
H
Segawa
Shin-ichi
S
Dobson
Christopher M
CM
Schwalbe
Harald
H
D000818:Animals; D002645:Chickens; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D020407:Internet; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D014867:Water
D004220:Disulfides; D014867:Water; D014364:Tryptophan; D014508:Urea; D009113:Muramidase