Genetically incorporated non-canonical amino acids (NCAAs) offer a wide range of chemistries not available with the 20 proteogenic amino acids. Several NCAAs can act as fluorescent probes that report on protein structure, dynamics, localization, and/or molecular interactions. A major benefit of using an NCAA over current fluorescent technologies is that these probes are similar in size to canonical amino acids allowing them to be rationally designed into the core of a protein without major modifications to the target protein. This provides the opportunity for fluorescent protein-based biosensor designs that can be tuned to the chiral nature of a protein/ligand complex, the conformational variability of protein structures, or the core/solvated regions of a protein. However, the limitation to rational design with NCAAs is the lack of structural data on the interactions governing the spectral changes in protein environments. We performed both absorption and fluorescence spectroscopy on seven variants of streptavidin containing the fluorescent NCAA L-(7-hydroxycoumarin-4yl) ethylglycine (7-HCAA). Of the five streptavidin variants that showed a response to biotin binding, three distinct spectral signatures were identified and structurally characterized with x-ray crystallography. In addition to characterization, we use the interactions identified in structural studies as input for computational protein design. We generated several calmodulin-based variants that were predicted to increase in fluorescence upon calcium binding. Of the five experimentally characterized designs, two showed such increases upon the addition of calcium. Finally, in order to better understand the interactions we have characterized and to conceptualize additional interactions we believe could occur between 7-HCAA and a protein environment, a set of TD-DFT calculations has been performed and analyzed. Together, these techniques could find widespread use in developing rationally designed biosensors or in the studying of protein structure/function.

Next-generation protein-based fluorescent sensors

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.