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The goal of this project is to understand how fundamental chemical interactions that occur within a biomolecule (e.g., protein), between biomolecules, or between a biomolecule and an inorganic surface drive the various molecular processes needed to sustain life and how these interactions in turn affect the environment and human health. To achieve this goal we have the following two objectives: <OL> <LI> Develop novel methods of fluorescent and scanning probe microscopy to characterize organic-inorganic interactions on a single-cell and single-molecule level. <LI> Isolate, identify, and characterize microbial (e.g., magnetotactic bacteria, Shewanella, Staphylococcus) and viral (e.g., prions from chronic wasting disease, scrapie) proteins that are involved in organic-inorganic processes such as dissimilatory metal reduction, biomineralization of nanominerals, development of environmental and/or pathogenic biofilms, and environmental transmission of microbial- or viral-based diseases.
NON-TECHNICAL SUMMARY: <BR> 1. By developing new imaging capabilities that combines the nanometer-scale resolving power of the scanning probe microscope with the chemical identification capabilities of a fluorescence microscope we will open up interesting possibilities for the use of this instrument in various fields of research, including material, geological, and life sciences. <BR>2. Understanding how soil microorganisms utilize specific membrane-proteins for metal-oxide reduction and biomineralization will give us a greater insight into how these processes can affect the mobility of pollutants (e.g., uranium, chromium) in the subsurface environment. Furthermore, by understanding the molecular mechanism that microbes direct the synthesis of mineral nanoparticles we can develop an important paradigm for bioinspired materials synthesis that will provide enormous insight into the strategies of controlled crystal synthesis used by other organisms, including multi-cellular organisms, and provide the basis for bio-controlled approaches to synthesize tailor-made inorganic nanostructures for applications across a diverse span of technologies. <BR>3. Determining the mechanism(s) by which microorganisms or viruses use proteins to recognize specific solid substrates to produce pathogenic biofilms on the solid support or bind to a mineral substrate in a manner that promotes the infectious nature of the microbe or virus. By understanding this process we will be able to help prevent environmental outbreaks with human or economic consequences.
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APPROACH: <BR>Objective 1 - An integrated atomic force microscope (AFM; Veeco/Digital Instruments; Bioscope AFM and NanoScope IV controller) / confocal laser scanning microscope (Zeiss; Axiovert 200M and LSM 510 META) will be used to collect simultaneous fluorescence and force measurements between inorganic substrates and biomolecules or cells. This instrument is located in Dr. Lower's laboratory at The Ohio State University Columbus, Ohio. Microbial cells will be located on a cover slip by using transmitted light and a 100X/1.45 N.A. objective lens on the confocal laser scanning microscope and an AFM cantilever will be positioned directly over a monolayer patch of cells (or uniform layer of biomolecules) prior to collecting force data. Fluorescence images will be collected by exciting cells with a 458/488 nm Argon laser and collecting the emission on a photodiode detector after passing the emitted light through a filter. Briefly, the tip of a cantilever will be brought into contact with a cell or biomolecule (i.e., "approach" force curves) and then pulled from the substrate surface (i.e., "retraction" force curves). The raw-data will be collected as the output of the photodiode detector (which is directly proportional to the deflection of the cantilever) relative to the position of the tip, which is translated by a piezoelectric scanner. These raw data are plotted as so-called "voltage-displacement" curves that may then be converted into "force-distance" curves and analyzed with SPIP (Image Metrology) and Igor Pro (WaveMetrics) software. For direct imaging, scanning tunneling microscopy, tapping mode or phase contrast mode will be used to collect detailed image (i.e., nanometer-scale resolution) of the samples. In addition to imaging the protein-mineral sample using single-molecule recognition force microscopy, we will also use AFM and STM to collect force and tunneling measurements between single prion molecules and mineral/soil substrates. <BR><BR>Objective 2 - Microbes will be grown under a variety of conditions (e.g., aerobic, anaerobic) and proteins will be purified from the cells for Objective 1 or for identification and characterization. By growing the microorganisms under different conditions and subsequently purifying membrane-associated proteins from these cells we will be able to identify proteins that are involved in specific biogeochemical processes (e.g., biomineralization) and understand the molecular mechanisms that these microbes are utilizing to live in a particular environment and, in turn, examine the environmental and societal consequences of their metabolic processes. Soluble cytoplasmic and peripheral proteins are first removed using the sodium carbonate solution and insoluble material collected by centrifugation. The resulting pellet, now enriched with membrane proteins, will be solubilized using a solution of much stronger solubilization reagents allowing for subsequent resolution of the membrane proteins using 2D gel electrophoresis. Protein spots were excised, in-gel tryptic digestion and mass spectrometry (MS) will be performed. Protein identification will be preformed using both automated and manual database searches.