Function and structure of cecropin A / Loraine Silvestro.

Silvestro, Loraine.
x, 146 p. : ill. ; 29 cm.

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Medical subjects:
Pharmacological Sciences.
Dissertations, Academic.
Local subjects:
Penn dissertations -- Pharmacological sciences. (search)
Pharmacological sciences -- Penn dissertations. (search)
Cecropin A is a naturally occurring, linear, cationic, 37-residue antimicrobial peptide. The precise mechanism by which it kills bacteria is not known, but its site of action is believed to be the cell membrane. To investigate the nature of its membrane activity, the interactions of cecropin A with synthetic lipid vesicles, bacteria and planar lipid membranes were investigated using various techniques.
In vesicles, the action of cecropin A was concentration dependent, forming ion channels at low peptide to lipid ratios, and "pores" large enough to pass probe molecules at higher peptide to lipid ratios. Cecropin A was equally effective on anionic and neutral vesicles, even though anionic vesicles bound more peptide. Cholesterol did not prevent dissipation of ion gradients by low concentrations of peptide, but did inhibit release of encapsulated probe by high concentrations of peptide. The findings in lipid vesicles suggest neither cholesterol nor anionic lipids determine cecropin A's selective action against bacteria.
The ability of cecropin A to permeabilize and depolarize membranes of bacteria was compared to its bactericidal activity. Results indicated that differences in the concentration dependence of membrane permeabilization and depolarization seen in synthetic vesicles were not manifested in bacteria. The concentration dependence of both phenomena correlate with bactericidal activity, suggesting that the bactericidal mechanism of cecropin A is related to membrane permeabilization.
To better understand the peptide activity in membranes, internal reflection infrared spectroscopy was used to characterize the conformation of cecropin A as it approached, adsorbed onto, and inserted into lipid membranes. Results indicate that cecropin A folds into final structure while superficially adsorbed to a membrane surface. Interactions with deeper hydrophobic regions of the bilayer appear to be unnecessary for folding. Its conformation is predominantly alpha-helical with some beta structure. The longitudinal axis of the helical structure, and the transverse axes of any beta structure, are preferentially oriented parallel to the membrane surface. These findings suggest a mechanism of action against vesicles that involves cooperative action of two or more peptides to produce a transmembrane defect.
Supervisor: Paul H. Axelsen.
Thesis (Ph.D. in Pharmacological Sciences) -- University of Pennsylvania, 2000.
Includes bibliographical references.
Local notes:
University Microfilms order no.: 9965567.
Axelsen, Paul H., advisor.
University of Pennsylvania.