| dc.description.abstract |
<p>Biosensors incorporating proteins as molecular recognition elements for analytes
are used in clinical diagnostics, as biological research tools, and to detect chemical
threats and pollutants. This work describes the application of protein engineering
techniques to address three aspects in the design of protein-based biosensors; the
transduction of binding into an observable, the manipulation of affinities, and the
diversification of specificities. The periplasmic glucose-binding protein from the
hyperthermophile Thermotoga maritima (tmGBP) was fused with green fluorescent protein
variants to construct a fluorescent ratiometric sensor that is sufficiently robust
to detect glucose up to 67°C. Ligand-binding affinities of tmGBP were changed by
altering a C-terminal helical domain that tunes ligand binding affinity through conformational
coupling effects. This method was extended to the Escherichia coli arabinose-binding
protein. Computational design techniques were used to diversify the specificity of
the E. coli maltose-binding protein (ecMBP) to bind ibuprofen, a non-steroidal antiinflammatory
drug. These designs ranged in affinity from 0.24 to 0.8 mM and function as reagentless
fluorescent sensors. The ligand affinities of ecMBP are tuned by complex interactions
that control conformational coupling. These experiments demonstrate that long-range
conformational effects as well as molecular recognition interactions need to be considered
in the design of high-affinity receptors.</p>
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