## LSU Historical Dissertations and Theses

#### Title

Solution Conformation of De Novo Antimicrobial Peptides.

1996

Dissertation

#### Degree Name

Doctor of Philosophy (PhD)

Chemistry

Mary D. Barkley

#### Abstract

De novo antimicrobial peptides were designed to help study the necessary conformational properties of biologically active peptides. One set of peptides are: (KLAKKLA)$\rm\sb{n}$, (KLAKLAK)$\rm\sb{n}$, (n = 1, 2, 3), (KALKALK)$\sb3$, (KLGKKLG)$\rm\sb{n}$, and (KAAKKAA)$\rm\sb{n}$ (n = 2, 3), while the second set have phenylalanine substituted for leucine, (KFAKFAK)$\sb3$ and (KFAKKFA)$\rm\sb{n}$, (n = 3, 4). These peptides were designed to be perfectly amphipathic in helical conformations. Circular dichroism was used to study their secondary structure in aqueous solution, sodium dodecylsulfate micelles, and phospholipid vesicles. Peptide antibacterial activity was tested against E. coli, P. aeruginosa, and S. aureus. Peptide cytotoxicity was tested against a mammalian cell line, 3T3 mouse fibroblasts. Our results demonstrate that the propensity to $\alpha$-helical conformation of the peptides in amphipathic media is proportional to their cytotoxicity. Sedimentation equilibrium was used to determine the aggregation state and equilibrium constant of the self-association of the peptides in aqueous solution. The thermodynamics of these associations were calculated from the temperature dependence of the association constants. With the exception of (KLGKKLG)$\sb3$ which does not self-associate, the other peptides are in a monomer-tetramer equilibrium. The peptides aggregate through the interaction of their hydrophobic residues. This was detected using 1-Anilio-naphthalene-8-sulfonate fluorescence. The phenylalanine peptides are 1.0-1.5 kcal/mol less stable than the equivalent leucine peptides. To correlate the aggregation and biological activity, carboxyfluorescein labeled peptides were used to determine the aggregation state of CF-(KLAKKLA)$\sb3$ and CF-(KLGKKLG)$\sb3$ in DPC micelles. CF-(KLAKKLA)$\sb3$ forms dimers while CF-(KLGKKLG)$\sb3$ is a monomer. These findings suggest that aggregation may contribute to the selective cytotoxicity of the peptides.

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