Continuous discoveries in bioengineering and more efficient molecular biology methods have allowed scientists to create new designer biomolecules with unique and distinct properties. In that regard, bio- nanotechnology and nanoscale analysis are becoming increasingly prevalent, raising the demand for techniques with high sensitivity that can detect biomolecules in biological samples. Bioluminescent photoproteins, such as aequorin, possess great potential to provide with a solution to this new challenge because they can be detected at low concentrations, and have different emission wavelengths depending on the protein variant used, a property that can be exploited in multiplex analysis. We now propose to prepare new aequorin variants to broaden the scope of their use in bioanalysis, thus allowing for detection of biomolecules that are not detectable by other technologies. Protein molecular switches with optical properties are another type of designer biomolecules that, in the presence of a target ligand, demonstrate an altered response manifested by an "on/off" signal. These molecules can be useful in a variety of applications, such as in the development of nanosensors for in vitro and in vivo detection. To that end, we plan to design and develop bioluminescent molecular switches that incorporate the recognition properties of binding proteins with the bioluminescence afforded by the aequorin variants. The hypotheses formulated for the proposed work are based on knowledge gained during our current funding period, and investigate the use of a series of computational and synthetic approaches along with genetic engineering strategies targeting the alteration of the electronic environment of the chromophore that should lead to new bioluminescent proteins and molecular switches with a wide range of spectral properties. These photoproteins will be employed in the development of assays for important biomolecules. Finally, we will investigate the use of the newly prepared bioluminescent molecular switches in the multiplex analysis of biomolecules and in the simultaneous analysis of molecules in single cells. We anticipate that the new photoproteins will provide with new enabling technologies for in vitro and in vivo biosensing, imaging, and multiplex analysis that have a number of advantages over existing methods.
The increasing importance of nanoscale analysis has raised the demand for highly sensitive systems that can detect biomolecules in biological samples. Bioluminescent photoproteins, such as aequorin, possess great potential to provide a solution to this new challenge because they can be detected at very low concentrations in physiological fluids. In that regard, we plan to design and prepare genetically modified photoproteins that form the basis of enabling technologies for the detection of relevant biomolecules and panels of biomarkers for in vitro and in vivo applications.
|Qu, Xiaoge; Rowe, Laura; Dikici, Emre et al. (2014) Aequorin mutants with increased thermostability. Anal Bioanal Chem 406:5639-43|
|Andreev, Victor P; Head, Trajen; Johnson, Neil et al. (2013) Discrete event simulation model of sudden cardiac death predicts high impact of preventive interventions. Sci Rep 3:1771|
|Hamorsky, Krystal Teasley; Ensor, Charles Mark; Pasini, Patrizia et al. (2012) A protein switch sensing system for the quantification of sulfate. Anal Biochem 421:172-80|
|Scott, Daniel; Hamorsky, Krystal Teasley; Ensor, C Mark et al. (2011) Cyclic AMP receptor protein-aequorin molecular switch for cyclic AMP. Bioconjug Chem 22:475-81|
|Trajkovic, Sanja; Zhang, Xiaoning; Daunert, Sylvia et al. (2011) Atomic force microscopy study of the conformational change in immobilized calmodulin. Langmuir 27:10793-9|
|Rowe, Laura; Ensor, Mark; Mehl, Ryan et al. (2010) Modulating the bioluminescence emission of photoproteins by in vivo site-directed incorporation of non-natural amino acids. ACS Chem Biol 5:455-60|
|Wei, Yinan; Daunert, Sylvia (2010) Enabling technologies in discovery: the 2009 Nobel Prize and its implications in antibiotic design. Anal Bioanal Chem 396:1623-6|
|Rowe, Laura; Dikici, Emre; Daunert, Sylvia (2009) Engineering Bioluminescent Proteins: Expanding their Analytical Potential. Anal Chem :|
|Doleman, Leslie; Davies, Logan; Rowe, Laura et al. (2007) Bioluminescence DNA hybridization assay for Plasmodium falciparum based on the photoprotein aequorin. Anal Chem 79:4149-53|