Alison
A. Smith
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Mailing
Address: 4044 Derring Hall Blacksburg, Virginia 24061 USA Office: 5086 Derring Hall Office Phone: 540.231.4147 Fax: 540.231.3386 alisons@vt.edu |
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Research
interests: |
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| Organisms that inhabit soils have a heightened risk for toxicity resulting from accumulations of heavy metals. Many plant species have developed the ability to control the uptake of these metals as a defense mechanism in order to maintain homeostasis. The levels of essential metals must be regulated to achieve proper concentrations of nutrients while harmful metals must be sequestered from the physiological systems of plants. However, the specific mechanisms underlying metal balance and detoxification remain largely unknown (Callahan et al., 2005, J. Biol. Inorg. Chem., 11: 2-12). The identification of metal-binding proteins, such as CdI19 from Arabidopsis thaliana (Suzuki et. al, 2001, Plant Cell Environ., 24: 1177-1189), offers the opportunity to develop a new type of biosensor to further study the molecular mechanisms of heavy metal-binding. One possibility is to modify a class of well-established biosensors, termed "cameleons" (Miyawaki et al., 1997, Nature, 388: 882-887). This construct uses the differences in excitation and emission spectra of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to signal changes in protein structure caused by a particular environmental condition. Conformational changes bring CFP and YFP into close proximity, allowing fluorescent resonance energy transfer (FRET) to occur between the indicators. These induced changes are detected and quantified by confocal microscopy. FRET can be difficult to monitor experimentally because it requires a specific distance between two fluorescent proteins. Another proposed method to detect the heavy metal binding abilities of proteins from A. thaliana is based on a recently developed technique, bimolecular fluorescence complementation (BiFC). BiFC uses two non-fluorescent fragments of YFP to monitor protein-protein interactions. Two fragments are brought together by positive interactions of the proteins to which they are fused, creating a functional fluorescence that can be monitored by a simple spectral fluorimeter. If either approach is successful, it will then be possible to investigate the binding abilities of CdI19 to other heavy metals. It may also be possible to insert cDNA libraries into these constructs and screen for novel proteins from other hyperaccumulating plants. | ||
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