Two-dimensional1H NMR study of recombinant insect defensin A in water: Resonance assignments, secondary structure and global folding

A 500 MHz 2D1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities,3JNH-αH coupling constants as well as1H/2H exchange rates and Δδ/ΔT temperature coefficients of NH protons strongly support the existence of an α-helix (residues 14–24) and of an antiparallel β-sheet (residues 27–40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from3JNH-αH coupling constants, (iii) 12 hydrogen bonds mostly deduced from1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a β-sheet is linked to an α-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.