Projects and Grants per year
Grants and Contracts Details
Description
Bioluminescent photoproteins afford high sensitivity and have been employed as labels in
bioanalysis. We propose to further expand the applications of these photoproteins by
modifying them to possess unique bioluminescence properties. One of the goals of this
work is to alter the electronic and H-bonding network within the chromophore-binding
pocket of these photoproteins in order to shift their emission wavelengths. This well be
achieved by incorporation of non-natural amino acids into the aequorin structure and by
performing site-directed mutagenesis. The resulting proteins will be characterized in
terms of their activity and structure-function relationships. We also propose to mimic the
natural phenomenon that occurs in the jellyfish Aequorea victoria where transfer of
energy from aequorin to GFP results in the emission of green light. For that, we propose
to prepare protein-based "artificial jellyfish" by attaching a fluorophore to unique sites on
aequorin close to the coelenterazine binding pocket. This will allow transfer of energy
from aequorin to the fluorophore, thus, shifting the wavelength of emission of the protein.
"Molecular switches" that can be "turned on" in the presence of a target analyte will be
prepared by constructing hybrid proteins between aequorin variants and a binding protein
for a specific analyte. The hybrid proteins will be constructed by inserting the gene of
the binding protein into the gene of aequorin. The conformational changes that occur in
the binding protein upon ligand binding will bring the two parts of aequorin together,
allowing for emission of bioluminescence. In addition, "molecular switches" will be
constructed by preparing fusion proteins of a dissected aequorin and genes that code for a
pair of polypeptides that can form a leucine zipper. The leucine zipper allows for
aequorin re-assembly and bioluminescence emission. When target DNA is present, the
leucine zipper is pulled apart, aequorin is disassembled, with the subsequent loss of light.
Application of the above modified photoproteins in bioanalysis will be demonstrated by
developing highly sensitive assays for panels of analytes that are important in disease
diagnosis. Furthermore, these assays based on aequorin variant with different emission
wavelengths will be incorporated into a micro centrifugal microfluidic platform, which
should result in multiplex analysis with applications in point-of-care diagnostics.
Status | Finished |
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Effective start/end date | 1/1/05 → 3/31/08 |
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Projects
- 1 Finished
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Modified Photoproteins as Labels and Molecular Switches in Bioanalysis
Daunert, S. (PI)
4/1/04 → 1/14/09
Project: Research project