 Hi, I'm Susanna Leong. In this work, we investigated the influence of amino acid composition of a short antimicrobial peptide on the peptide's antimicrobial properties, salt tolerance as well as biocompatibility behavior. So the focus of this study is to essentially try to understand the balance between peptide hydrophobicity and charge on those properties. The motivation of this study is to engineer peptides that can eventually be used as antimicrobial bullets for a variety of in vitro applications including coating agents. Antimicrobial peptides are fascinating molecules that show great potential as second generation antibiotics. They kill microbes by targeting their membranes, a core structure that is difficult to evolve quickly, and this reduces the risks of antibiotic resistance development compared to conventional antibiotics. For maximum efficacy, antimicrobial peptides or AMPs in short must possess broad-spectrum antimicrobial activity, be biocompatible and salt tolerant. Unfortunately, natural AMPs do not possess all these attributes, but this can be resolved by rational design of these peptides, guided by good understanding of peptide structure function, which forms the basis of our study. We thought that AMPs that have a good balance of hydrophobicity and charge would have made brilliant candidates and went ahead to investigate the influence of charge and hydrophobicity on the activity of tryptophan and arginine-rich decamer peptides. The parent peptide from which we engineered the TANMER peptide derivatives is the C-terminus of a human beta-defending 28 variant, which our group previously engineered for salt-resistant properties. Systematic amino acid substitution of the 10 residue peptides with tryptophan and arginine residues was performed to determine the optimal hydrophobic-to-charge ratio that is required for broad-spectrum and salt-resistant antibacterial activity. Out of the six variants generated, two D5 and D6 showed superior antimicrobial activity with minimum inhibitory concentrations at sub-micromolar concentration towards E. coli, Staphylococcus areas and Pseudomonas aeruginosa. C-terminal amination of the peptides further improved the minimum inhibitory concentration or MIC values of the decamers, which maintained good salt-resistance and biocompatibility properties. Having obtained two fantastic peptide variants, we proceeded to characterize their membrane-interacting behavior in an attempt to improve structure-function knowledge of these molecules. NMR studies of D5 in the presence of SDS, which mimics the bacterium membrane, show that backbone conformations of both D5 and D6 in SDS micelles are predominantly extended, with significant side-chain-side-chain proximities, which potentially indicate cation-py type interactions. Such cation-py interactions are thought to be critical for broad-spectrum antimicrobial activities of these arginine tryptophan-rich AMPs. We also studied the peptide localization in SDS micelles by comparing the relative quenching of the buried tryptophan fluorophore in the presence of two nitrocyte spin labels, which affects the fluorescence intensity differentially based on the localization of the peptide in the lipid milieu. Both D5 and D6 were found to be localized at the lipid-water interface in the SDS micelles, and therefore shows a possible membrane disruption mechanism through detergent-like activity. Isothermal calorie-metry measurements demonstrate the high binding affinities and the negative enthalpy of the peptides upon binding to negatively charged POPG-LUVs, which suggests a potential dominant role for electrostatic interactions in the peptide's preferential binding to anionic bacterial membranes. The fundamental characterization of the engine and decimus provided through this study has improved our structure function understanding of short arginine tryptophan-rich AMPs. Now, the outcome of this work will certainly pave the way for future de novo design studies of short potent AMPs for biomedical or therapeutic applications. My group is currently working towards tethering these peptides on biomedically relevant surfaces. So, I hope that you found our work interesting and useful. Thank you very much.