(591e) Cysteine-Containing Antimicrobial Peptides: The Effects of Cysteine Oxidation on Solution Behavior and Antimicrobial Performance

As drug-resistant bacteria become more prevalent, causing increased health and economic stress, the development of novel antimicrobial agents is critical. We found a human chemokine-derived peptide D8 (_D-RTVRCTCI) to show high activity against a broad spectrum of bacteria as well as high proteolytic stability. To understand the role that each amino acid plays in this activity we replaced residues one-by-one with _D-alanine and tested the bactericidal activity of the 8 resulting peptides. We found that replacing arginine, isoleucine, and valine reduced activity, likely due to reductions in the cationic and hydrophobic character of the peptide. Yet, the substitution of cysteine with _D-alanine or even serine also resulted in a loss of activity, suggesting that cysteine plays a key role in the antimicrobial effects exhibited by this peptide. Since cysteine residues are known to be oxidizable and affect protein/peptide structure and function, we are interested in understanding how the number and oxidation state of cysteines impact the properties of peptide D8. We synthesized 3 D8-derived peptides with acetamidomethyl (Acm) caps, which prevent oxidation of the thiol groups, on either or both cysteine thiol groups using Fmoc-chemistry. We found capping both cysteines with Acm groups decreased antimicrobial activity. Since cysteine residues can become oxidized, altering the structure and properties of cysteine-containing peptides, we sought to examine the role of cysteine oxidation on the solution properties and performance of these D8 peptides. Specifically, this talk will cover our investigation of D8 peptides with one Acm cap on either the most N-terminal (5^th residue) or the most C-terminal (7^th residue) cysteine. We hypothesize that the cysteine at position 5, next to arginine, is more reactive than the other and will affect the oxidation rate and solution behavior of the resulting materials more. To monitor oxidation, we use high-performance liquid chromatography (HPLC), diffusion order spectroscopy (DOSY), and Ellman’s assay. To determine the structures of the oxidized peptides, we use matrix-assist laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy. This presentation will also discuss the size, zeta potential, and morphology of the investigational peptides in different buffers at various concentrations, as characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Understanding the role of cysteines and their oxidation in the solution behavior and antimicrobial performance of D8 will advance the molecular design of cysteine-containing peptides as novel antimicrobial agents.