Abstract:
Fluorescent Proteins are intriguing molecules that are ubiquitous tools for biological research. My thesis investigates the relationship between engineering improved versions and fundamental physical chemistry principles that drive the molecular design of brighter and more photostable variants of existing fluorescent proteins. My talk will primarily focus on designing brighter variants of two red-fluorescent proteins, FusionRed and mCherry, and the consequent physical investigations that led to increments in brightness in these molecules. To achieve this, droplet-based and single stream microfluidic sorters that discriminate members of a cell-based mutagenesis library of variants on their excited state lifetime and fluorescence intensity were developed and utilized. These lifetime-based selections led to variants with greater than 3-fold higher values of fluorescence quantum yield in comparison to their progenitors. Finally, the pathways leading to excited state depopulation after photoexcitation, which govern observables like fluorescence quantum yield, fluorescence brightness, reversible and permanent photobleaching, and blinking at the single molecule level were investigated. I will focus on the progress and the many knowledge gaps in understanding the interwoven relationships of observed photophysics in fluorescent proteins, and suggest ways in which one might further examine the physical basis of brightness and photostability in these molecules.
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The talk will be held in JILA (Main Campus, CU Boulder). Room X317 (3rd Floor, X-Wing; by the h-bar) will be the venue. This talk will be held in a hybrid format to accommodate anyone who is uncomfortable or unable to attend an in-person defense.
Please be seated in the room by 2 PM and kindly mute your audio if attending on zoom.
Zoom info and passcode included in the email announcement.