2-DIMENSIONAL H-1-NMR STUDY OF RECOMBINANT INSECT DEFENSIN-A IN WATER - RESONANCE ASSIGNMENTS, SECONDARY STRUCTURE AND GLOBAL FOLDING

A 500 MHz 2D H-1 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, 3J(NH-alpha-H) coupling constants as well as H-1/H-2 exchange rates and DELTA-delta/DELTA-T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-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 from 3J(NH-alpha-H) coupling constants, (iii) 12 hydrogen bonds mostly deduced from H-1/H-2 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 beta-sheet is linked to an alpha-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.