Chapter 1 NMR of Antimicrobial Peptides

Abstract

Abstract The continuing increase in the resistance of pathogenic bacteria to conventional antibiotics has led to the emergence of new strains that are often referred to as ‘superbugs’. There is a serious need for new anti-infective therapies as clinicians can no longer effectively combat these bacterial pathogens. In the search for new and effective antibiotic compounds, antimicrobial peptides, which are part of a larger group of host defence peptides and proteins, have emerged as a potential class of agents that may stem the tide of antibiotic resistance. However, the mechanism of action of antimicrobial peptides is currently poorly understood. Widely different and complex models for the microbicidal action of antimicrobial peptides have been proposed. Consequently, in order to optimize the design of new synthetic antimicrobial peptides, each peptide must be examined on its own to determine which factors are most responsible for its bactericidal or bacteriostatic activity. Nuclear magnetic resonance (NMR) spectroscopy has become the tool of choice for determining the structures of the antimicrobial peptides and to characterize their interactions with their initial target, the negatively charged bacterial membrane. Moreover, interactions with zwitterionic eukaryotic membranes are also surveyed in an attempt to avoid host cytotoxicity of these peptides. Standard solution-state NMR techniques are now commonly used to determine the high-resolution structure of an antimicrobial peptide in aqueous solution, in detergent micelles or in membrane mimetic organic solvents. Solid-state NMR is increasingly being used to examine the orientation of the peptide in phospholipid bilayers as well as the oligomeric state of the peptide; it can also be used to characterize the changes in biological membranes that are induced upon peptide binding. Through the incorporation of 15 N, 13 C and 2 H nuclei into antimicrobial peptides by recombinant fusion-protein expression techniques, as well as advances in NMR technology and experimental procedures, antimicrobial peptides can now be studied in large membrane mimetic vesicles which more closely resemble biological membrane bilayers in vivo . This review paper will discuss many of the solution and solid-state NMR experiments that have been used to characterize antimicrobial peptides and that have provided researchers with unique insights into the diverse mechanisms of action of this class of biomolecules.