This is the area of research that I have worked on continuously since joining UTK. I began working on this area during my NSF Postdoctoral Research Fellowship in the lab of Dr. Kenneth Keegstra. Although I formally began working on this as a post-doctoral fellow, it was a natural extension of my graduate work, which investigated the "Structure, Function and Assembly of Photosynthetic Electron Transport Complexes." Since many of the subunits found within these complexes are nuclear-encoded, investigating how they are targeted and translocated into chloroplasts was an obvious and related area to address after completing my Ph.D. Although we are still uncertain how this process takes place, it is now clear that the vast majority of chloroplast-targeted proteins are encoded as a larger precursor that contains an N-terminal protein extension known as a transit peptide. Although computer programs can predict these sequences, the precise function of these peptides is still poorly understood. It has been my goal to dissect the protein targeting/translocation process down into discreet steps that may therefore allow a systematic attempt to "decode" how transit peptides function. To enable this effort we have focused our attention on a well-characterized precursor and its associated transit peptide, prSSU (precursor to the small subunit of Rubisco). During this pursuit my laboratory developed tools to determine the many sequential or even concurrent interactions that transit peptides perform: 1) transit peptide-molecular chaperone interaction, 2) transit peptide-chloroplast lipid interactions, 3) transit peptide-mature domain interactions, 4) transit peptide interactions with the Toc receptors, Toc34 and Toc159, 5) transit peptide interactions with Tic components and PIRAC, 6) transit peptide recognition with the stromal processing peptidase, and finally 7) transit peptide degradation by the presequence degrading protease, preP1/2. My lab has now published several papers in each of these areas. We have begun to develop a working model for how at least one transit peptide can be designed to perform these multiple interactions. Future work will explore the dynamics of the nanoscale molecular machines using a combination of singlemolecule. Currently supported by NSF Program in Cell Biology.
Photonics and Applied Photosynthesis:
Integration of Photosynthetic Complexes into Hydrogen-evolving Nanoparticles and Solid-state Photovoltaic Devices
Although I have worked investigating the light reactions of photosynthesis since undergraduate days at UCSC, I had largely shifted my interest from photosynthesis to chloroplast biogenesis and chloroplast protein import during my post-doc and as an assistant professor. However, from the very beginning I have always held that photosynthesis held the secret for a viable means of solar energy production. This interest and conviction led me several years ago to initiate collaboration with Dr. Elias Greenbaum to explore the use of isolated photosynthetic complexes in applied photosynthesis. In particular, we began to use the isolated PSI complexes which I had worked extensively with as a graduate student as sources of high-energy electron for hydrogen production. This project involved the platinization of the PSI complex under anaerobic conditions. Following platinization we were able to show that these PSI nanoparticles were capable of producing hydrogen upon illumination. These particles were shown to be highly robust catalysts that could take electrons from cyt. c/plastocyanin and evolve hydrogen on their acceptor side. This was work initially conducted with a talented undergraduate, Jennifer Millsaps, who functioned as a conduit from my lab to the Greenbaum lab at ORNL. This collaboration is ongoing and is now the basis of a collaborative proposal to the DOE for their Solar Energy. During this collaboration I was introduced to Dr. Marc Baldo an Assistant Professor of Electrical Engineering at MIT. Dr. Baldo, Elias Greenbaum, and I worked together on a Phase I grant from DARPA to test if PSI could be made integrated into solid-state devices. From this initial interaction, I have continued my collaboration with Dr. Baldo and we have now published three papers. This work is currently being funded through a $1.8M NSF NIRT (Nanoscience Interdisciplinary Research Team) grant. I am the PI on this grant and coordinate this highly collaborative project involving my role in microbiology, molecular biology and membrane biochemistry and two engineers from MIT (Dr. M. Baldo and Dr. S. Zhang, Biomedical Engineering). Together we have demonstrated the ability to take isolated photosynthetic reaction centers from plants or bacteria and to integrate them into a solid-state device using organic semiconductors. These devices have the ability to convert absorbed light into a photocurrent with an internal efficiency of ~12%. We are now designing new devices that incorporate large phycobilosomes from marine cyanobacteria as massive light harvesting antennae with reaction centers isolated from thermophilic cyanobacteria. The successful integration of a supramolecular antennae, a thermostable reaction center, and efficient coupling via surface plasmons will allow us to built devices that are larger, more optically dense, have a high quantum efficient, and are more stable. Currently supported by NSF NIRT grant, NSF IGERT Program and NSF Program in Sustainable Science.
Nanoencapsulation of Antimicrobial Peptides
My early work with transit peptides indicated that one of their properties is that they are membrane active. This activity is reflected by both their ability to disrupt/lyse membranes with the correct lipid composition and also by the peptides ability to undergo a conformational shift to reflect an increase in alpha helical content. During this work I began working with another class of membrane active peptides, known as antimicrobial peptides such as magainin and nisin. This work was initiated by my interaction with a young biophysist, Jochen Weiss, in the Food Science and Technology in the institute of Agriculture at UTK. We actually developed two concurrent projects that involve in one form or another the interfacial properties of peptides and proteins. The first project was to investigate how physical treatments in the food industry such as high intensity ultrasound affects the surface activity of "model" food proteins such as BSA, β-lactoglobin, and lysosome. My role was to essentially use "soft" structural biology techniques such as CD, DSC, FTIR, native gel electrophoresis; surface hydrophobicity measurements using PRODAN/ANS fluorescence, and limited proteolysis. This work was the work of two M.S. students (Guzey and Gulersen) that we co-mentored and has lead to two recent publications with one manuscript still in preparation. A second project was the result of the formation of the Center of Excellence in Food Safety (CEFS) that was started at UTK in 2000. This was a collaborative project between Dr. Weiss, a food microbiologist (Dr. P. Michael Davidson), and myself. This collaborative work (supported by two CEFS Seed Grants and current USDA NRI grant) explores how novel nanoencapsulation of antimicrobial agents can be used to enhance the efficacy and longevity of food antimicrobial agents. In this project, my role was to develop and characterize novel liposome encapsulations that physically entrap the antimicrobial peptides or agents. Although many of these agents can be shown to be highly effective in preventing growth of Listeria, E. coli, and other foodborne, pathogens, their efficacy is often lost when introduced into the complex systems that are found in the food industry. This attenuation is often due to non-specific loss through absorption to food components. To prevent this we have developed new nanoscale encapsulations such as essential oil/surfactant micelles and liposomes. These particles can then be caused to release their content upon some physical/chemical trigger such as ultrasound, heat treatment, pH shift, and surfactant treatment. This work supported a joint post-doctoral fellow (Were), two M.S. students (Borland and Gaysinsky) and a Ph.D. student (Taylor) who will finish in the fall of 2006. This work has lead to seven manuscripts with several in preparation. Supported by USDA and Proctor & Gamble.
