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Laura M. S. Baker: I am a returned Peace Corps Volunteer from the Fiji Islands where I taught chemistry and mathematics at a rural school
Laura M. S. Baker: I am a returned Peace Corps Volunteer from the Fiji Islands where I taught chemistry and mathematics at a rural school.
Laura M. S. Baker
Undergraduate school: B.S. Chemistry, University of Puget Sound, Tacoma,WA
E-mail address: email@example.com
Advisor: Leslie B. Poole, Ph.D.
Supplement to CV
"I chose to attend WFU because I really liked how the small school atmosphere allowed students and professors to work together on a one-to-one basis. At other larger schools, I noticed the students had less access to their P.I., and there was more distance between students and professors, both socially and academically. At WFU, our biochemistry department is much more integrated, and all of the staff here promote an open door policy towards the students."
In the Poole lab, we study enzymatic systems which protect organisms from cellular damage caused by free radicals generated during oxidative stress. In particular, we're interested in alkyl hydroperoxide reductase (AhpC) systems (also referred to as Peroxiredoxins (Prx)) that are capable of converting toxic alkyl hydroperoxides formed in biological membranes by reactive oxygen species (ROS) to their corresponding alcohols. The cysteine-based AhpC peroxidase systems have been found in all biota, but of most interest to us are the AhpC systems in pathogenic bacteria. We primarily use spectroscopic techniques such as site-directed mutagenesis, UV/visible and fluorescence spectrophotometry, analytical ultracentrifugation, and rapid reaction kinetics to obtain information about the structure and function of these enzymes. Eventually these studies should help determine how these protective enzyme systems contribute to bacterial virulence and aid in the development of novel antibiotics.
For my thesis work, I have been studying the kinetics and mechanism of two bacterial Prx systems: 1) AhpC from Helicobacter pylori, the causative agent of stomach ulcers, type B gastritis, and gastric cancer, and 2) Thiol Peroxidase (Tpx) from Escherichia coli, a distantly related Prx homologue. For activity, both systems require a reducing system composed of Thioredoxin (Trx), Thioredoxin Reductase (TrxR), and NADPH (see Figure 1 below). ahpC knockouts in H. pylori created in collaboration with Paul Hoffman's group at Dalhousie University demonstrated that AhpC is required for H. pylori viability, the first example of an essential Prx. Using a combination of bisubstrate Dalziel kinetic analyses and AhpC-dependent assays, we were able to kinetically characterize both the H. pylori Trx1/TrxR reducing system and the interactions of AhpC with Trx and peroxides. The catalytic efficiency of H. pylori's TrxR/Trx1 system was one third that of the homologous E. coli system, in part due to the higher Km of H. pylori TrxR for Trx1 (22.6 µM versus 3.7 µM for the E. coli system). AhpC interacts with its substrates, Trx1 and hydroperoxides, according to two bimolecular reactions with infinite Vmax and Km values and with a second-order rate constant of 1-2 x 105 M-1s-1. indicating that AhpC plays an important role in cellular peroxide metabolism.
Studies on E. coli's Tpx have also emphasized kinetically characterizing the peroxidase's interaction with its reducing system. Unlike H. pylori's AhpC, Tpx does interact with Trx according to Michaelis-Menten kinetics (Km = 25 uM) and exhibits a higher catalytic efficiency of 3.0 x 106 M-1s-1. While AhpC interacts with its peroxides at similar rates, Tpx displays differential reactivity with various peroxides and prefers alkyl hydroperoxides such as CHP (Km = 9.0 uM) and fatty acid hydroperoxides over H2O2 (Km = 1730.91 uM), suggesting a more complex substrate binding site for Tpx than for AhpC. In addition, spectroscopic mechanistic studies were undertaken to determine the presence of a sulfenic acid intermediate, the oligomeric solution state, and the identification of the intra-subunit disulfide bond. Site-directed mutagenesis was used to create Cys to Ser mutants of Tpx's three Cys, and evaluation of these mutants has resolved that Cys60 is the essential, peroxidatic Cys, in contrast to previous reports that Cys94 was the required site.
Other: I am a returned Peace Corps Volunteer from the Fiji Islands where I taught chemistry and mathematics at a rural school. Please check out my links to Peace Corps-and Fiji-related websites.