Abstract 2463 Investigating the relationship between sequence, structure, and function of antimicrobial peptides in traditional and course-based research

The need for novel classes of antibiotics is urgent and growing as antibiotic-resistant bacterial strains continue to spread. Antimicrobial peptides (AMPs) are produced by a wide range of organisms, with diverse sequences, structures, and bacterial specificities. Most excitingly, AMPs are believed to kill bacteria primarily by physically disrupting the cellular membrane, which makes the rise of bacterial resistance more challenging. We are working with a class of AMPs, known as cecropins, that are produced by insects. Cecropins range from approximately 30-55 amino acids in length, lack disulfide bonds, and are primarily α-helical in structure. A better understanding of the relationship between the cecropin sequence and antimicrobial activity would better enable the development of antibiotic treatment. We are working to correlate small changes in the peptide sequences to differences in their biophysical properties and antimicrobial activity; I have also developed a course-based undergraduate research experience to design and test a broader pool of potential AMPs based on cecropin sequences. The design properties that we discover through the course will inform and inspire deeper studies performed in longer-term research projects. We are using solid-phase peptide synthesis to generate full-length and truncated cecropins based on the AMPs expressed by Drosophila melanogaster and Photinus pyralis. Once the peptides are synthesized and purified, we use a variety of techniques to study the biophysical properties of each AMP, including circular dichroism spectroscopy to analyze the secondary structure. We also determine the minimum inhibitory concentration (MIC) of each peptide against a variety of bacterial species. For the family of cecropins expressed by Drosophila melanogaster, we have discovered that a single amino acid mutation has a significant impact on the antibacterial properties of the AMPs. Cecropins are believed to have a helix-hinge-helix structure, and we found that replacing a glycine with a valine in the predicted hinge region (based on modeling using the I-TASSER structure prediction server) increases the efficacy of the AMPs. Further, using a novel cecropin isolated from the Photinus pyralis genome, we determined that the N-terminal fragment of the peptide is sufficient to prevent bacterial growth. I have also designed a course-based undergraduate research experience that allows students to design, synthesize, and test AMPs. Based on the results from my research lab and others in the literature, students in my course have designed peptides, based on the Drosophila melanogaster CecB sequence, that are truncated to include less than 16 amino acids and may also include amino acid mutations. My research group is working to better understand the relationship between the sequence and function of cecropins, and the results from undergraduates in the course are enabling us to much more rapidly develop and test interesting peptide leads. This work is supported by Bucknell University and the Scadden Research Fellowship.