Sensing the light: design of photoactivatable protein-protein interactions using the LOV2 domain Public Deposited
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- March 22, 2019
Lungu, Oana Izabela
- Affiliation: School of Medicine, Department of Biochemistry and Biophysics
- Photocontrol of protein-protein interactions is a powerful tool for precise spatial and temporal control of cell signaling networks. This dissertation has sought to expand the use of naturally occurring, light-sensitive proteins to photocontrol protein-protein interactions. This method allows for genetically encoded, reversible photoacitvation, or caging, of critical protein-protein interactions in a signaling network cascade. The small, globular GTPase protein Rac1 was caged through the use of the light-sensitive LOV2 domain as steric block to Rac1 effector binding in the dark. The resulting photoswitch which was created, PA-Rac, was able to induce membrane rufflng, protrusions, and directed cell movement upon blue light irradiation at the leading edge of mammalian cells. Its use has resulted in deeper understanding of the role of Rac1 in mammalian cell motility. Modeling and characterization of PA-Rac enabled caging of the related GTPase Cdc42 in a similar manner. The LOV2 domain was also used to create a photoactivatable peptide, LOV-ipaA. The photoswitch, which binds the adhesion protein vinculin, was designed by embedding a peptide sequence by into the J[alpha] helix of the LOV2 domain. As a model case, LOV-ipaA demonstrated the ability to alter the sequence of the LOV2 domain J[alpha] helix while maintaining its photoswitching capacity in order to introduce new functionality into the helix. LOV-ipaA light activated heterodimerization to vinculin was used as tool for light-induced protein expression in S. cerevisiae. This tool allowed for photoactivatable overexpression of the proteins Ste4 and Gpa1, which are critical components in regulation of the yeast pheromone mating response pathway. Through this work, signicant progress was achieved toward being able to photactivate two classes of proteins, and using those proteins as tools to manipulate biological systems.
- Date of publication
- May 2012
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- In Copyright
- ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics.
- Kuhlman, Brian
- Hahn, Klaus
This work has no parents.
|Sensing the light : design of photoactivatable protein-protein interactions using the LOV2 domain||2019-04-10||Public||