STRUCTURAL STUDIES OF IMPATIENS-BALSAMINA ANTIMICROBIAL PROTEIN (IB-AMP1)

Structural studies of Ib-AMP1, a small antimicrobial peptide derived f rom the seeds of Impatiens balsamina have been performed using circula r dichroism (CD) and two-dimensional proton nuclear magnetic resonance (H-1 NMR), This 20-residue peptide is highly basic with five arginine residues and contains four cysteines which form two intramolecular di sulfide bonds, CD results reveal that the peptide may include a beta-t urn but do not show evidence for either helical or beta-sheet structur e over a range of temperature and pH. Structural information from NMR was obtained in the form of proton-proton internuclear distances infer red from NOEs and dihedral angle restraints from spin-spin coupling co nstants, which were used for distance geometry calculations. Owing to the difficulty in obtaining the correct disulfide connectivities by ch emical methods, three separate calculations were performed; with no di sulfides and with the two possible alternate disulfide connectivities. Results from distance geometry calculations reveal that although the peptide is small, the cysteines constrain part of it to adopt a well-d efined main chain conformation. From residue 6 to 20, the backbone is well defined, whilst the N-terminal region, residues 1-5, has very few constraints and appears to be very flexible, In the defined core regi on, there are three beta-turns at residues 9-12, 10-13, and 12-15. The side chains show no strong interactions in the NMR spectra and are th erefore thought to adopt multiple conformations. Superposition of the structures generated shows that the peptide has two hydrophilic patche s which are at opposite ends of the molecule separated by a large hydr ophobic patch. Little is known about the mode of action of this protei n, but it is thought to interact with a membrane-bound receptor, and p ossible sites of interaction are discussed, The structures determined are compared with those of the a-conotoxins, which are also highly bas ic proteins with similar disulfide connectivities.