Heterologous expression of hen egg white lysozyme and resonance assignment of tryptophan side chains in its non-native states.
A new protocol is described for the isotope (15N and 13C,15N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. 1H,13C and 15N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the Cgamma resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.
Schlörb
Christian
C
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Marie-Curie-Strasse 11, D-60439, Frankfurt, Germany.
Ackermann
Katrin
K
Richter
Christian
C
Wirmer
Julia
J
Schwalbe
Harald
H
D000818:Animals; D002247:Carbon Isotopes; D002645:Chickens; D004926:Escherichia coli / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007211:Indoles / Q000737:chemistry; D008745:Methylation; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
D002247:Carbon Isotopes; D007211:Indoles; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; C030374:indole; D014364:Tryptophan; C421864:hen egg lysozyme; D009113:Muramidase