Preparation of isotopically labelled recombinant beta-defensin for NMR studies

Abstract

beta-Defensins are a family of cationic peptides that contain six invariant cysteine residues that form characteristic disulfide bonds between Cys(1)-Cys(5), Cys(2)-Cys(4) and Cys(3)-Cys(6). They have been shown to act as potent antimicrobial agents and chemokines. Human beta-defensin 2 (HBD2) was first isolated from psoriatic skin lesions and the structure of this peptide has been solved by X-ray crystallography and NMR spectroscopy both of which are consistent with a fold that contains an N-terminal alpha-helix and three antiparallel beta-strands. Here, we report the expression and purification of the first isotopically labelled beta-defensin ((15)N HBD2) with 100% incorporation of (15)N using a recombinant Escherichia coli method. Multidimensional NMR spectroscopy experiments: 2D (1)H-(15)N HSQC, 3D HSQC-TOCSY and 3D HSQC-NOESY allows for the assignment of resonances with no overlapping or ambiguous peaks. This isotopically labelled peptide is highly suitable for studying the interactions between HBD2 and a range of components from both the mammalian immune system and bacterial pathogens.