Antimicrobial Peptides:
AMPs are short, naturally occurring peptides that demonstrate broad spectrum bacteriocidal (killing bacteria) or bacteriostatic (stopping bacterial growth) properties. These peptides are found in most higher organisms and play a front-line role in the innate immune system. AMPs are typically 10-50 amino acids in length and are often unstructured until they interact with a target membrane, at which point they will often
adopt an alpha-helical structure. The
peptides are typically amphipathic helices with positively charged
amino acids on one face of the helix and hydrophobic amino acids
localizing to the opposite site of the helix. The mechanism by which
these peptides act is currently a hot debate. Many AMPs have been shown
to cause permeabilization of bacterial cell membranes which, in turn,
leads to the bacterium's inability to generate ATP and therefore
results in death. However, it has been demonstrated that these peptides
can cause killing or bacteriostasis at much lower levels than are
required for membrane permeabilization. Through transcriptional
profiling experiments it was shown that a variety of genes and aberrant
gene responses were a result of very low levels of exposure to AMPs. My research focuses on understanding what about these peptides make them (a) broad spectrum, (b) membrane permeabilizing, and (c) signaling activators. The initial phase of the investigation is focusing on the amino acid identities in the peptides. Naturally ocurring AMPs are limited to the 20 natural proteinogenic amino acids which limits and restricts the potential interactions. Changing the amino acid sequence to affect the physiochemical properties of the peptide as well as incorporating non-native amino acids through solid-phase synthesis methods will garner a more thorough understanding of which peptide properties play a role in which mechanisms of action. We use a combination of biochemical, bacteriological, and biophysical techniques to assay peptide activity. We are also interested in the lipid-mediated effects of peptide-membrane interactions. Specifically, since the lipid composition of the bacterial membrane is vastly different from that of a mammalian cell membrane we are investigating which lipid components are involved in each stage of the AMP interaction with the bacterial cell.
Transmembrane Peptides:
Over one-third (~33%) of all proteins in higher organisms are expected to be transmembrane or membrane associated proteins but approximately two-thirds (~67%) of all pharmaceutical drug-targets. These proteins can act as enzymes, channels, pores, pumps, and signal receptors. While we know that the most common secondary structural motif in transmembrane proteins is the alpha-helix, there is much less high resolution str
uctural information
available for
membrane proteins due to a variety of complicating experimental factros
stemming from their hydrophobicity and difficulty in high yield
expression methods. To overcome some of these restrictions, short
peptides that mimic a transmembrane helix have been very successfully
applied to study the behavior and dynamics of TM alpha helices. These
peptides can be model systems to understand the basics of behavior in
the membrane, or they can be sequence mimcs of natural TM sequences. My research in this area focuses on understanding what properties of these peptides are most important for determining their ability to form a TM helix. Natural TM helices have a variety of primary sequences, not all of which are hydrophobic residues. We'd like to learn more about how specific amino acids affect the equilibrium between transmembrane and non-transmembrane orientations. Specifically, we're interested in how the number and location in the helix of non-hydrophobic amino acids such as Asp, Lys, and other non proteinogenic amino acids (Dap, Orn, etc) affect this equilibrium. These amino acids can be charged or uncharged, depending on their location and the pH of the environment. We incorporate peptides containing these amino acids of interest into lipid vesicles to investigate their properties. How this ionization event affects their membrane topology is one of the basic questions in this area. The methodology we employ is based on physical methods (cysteine crosslinking, disulfide bond formation) and spectroscopic techniques including fluorescence spectroscopy, fluorescence quenching, and circular dichroism.