SOLUTION & SOLID-STATE NMR SPECTROSCOPY APPLIED TO THE INVESTIGATION OF ANTIMICROBIAL PEPTIDES ISOLATED FROM NATURAL SOURCES

            Nuclear Magnetic Resonance (NMR) is a powerful tool to investigate the mechanism of action of antimicrobial peptides. Whereas solution NMR affords the 3D structure of membrane-associated peptides in atomic resolution, the anisotropic parameters from solid-state NMR (ssNMR) give the topology of membrane-associated peptide.

            Our team has been working with antimicrobial peptides from different animal sources, so NMR has extensively been used to gain important insight into their mechanisms of action.

            The structural propensities of peptides are initially mapped by CD spectroscopy. Then 2D solution NMR experiments are performed to obtain restraints, which are used in simulated annealing protocols to give the peptide 3D structure. Based on the structure, specific amino acids are selected for labelling with ¹⁵N and ²H isotopes, and the respective labelled peptides are reconstituted into oriented phospholipid bilayers. ³¹P ssNMR experiments are performed to check on the membrane disturbance promoted by the peptides. The anisotropic ¹⁵N chemical shift and the¹H-¹⁵N dipolar splitting from ¹⁵N experiments, allied to the quadrupolar splitting from ²H ssNMR experiments, are used in a home-made program to afford the membrane alignment of the peptide.

            The models obtained from the combined use of the 3D peptide structure and its membrane topology offer valuable information on the peptide-membrane association. This included the partition of hydrophilic and hydrophobic residues, a clear depiction of the role of some aromatic residues into anchoring the peptide, as well as the partitioning and anchoring effects of some charged residues lying on the lipid bilayer interface. In some cases, hydrophilic or charged residues at the peptide C-terminus have been proved to be important on the control of the peptide-membrane alignment.

            NMR experiments performed with peptides encompassing similar primary structures have offered important insight on the role of specific amino acids, which has also allowed the design of new analogues. Experiments with peptide dimers in water and membrane media have given important insight on the combined action of the peptide chains. The combined use of NMR with other techniques has proved itself very important to the understating of the peptide-membrane binding.