Graduate Thesis Or Dissertation
 

Revealing Ultrafast Dynamics and Functional Basis of Potential Biomedical Tools from Calcium Sensing to Optogenetics

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/g158br373

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  • Photoactivated biomedical tools like fluorescent biosensors and optogenetic proteins have increased in popularity due to the precision targeting and activation used for in vivo applications. In nature, the initially discovered parent proteins exhibit properties such as fluorescence quantum yield (FQY), fluorescence color, and photoswitching dynamics that are unfavorable in mammalian applications. Tailoring these proteins for specific applications typically requires high-throughput random mutagenesis, which commonly lacks a photophysical understanding of the chromophore (or cofactor) interaction with the protein environment. Such “microscopic” interactions between the protein pocket and the embedded chromophore are crucial as removing or mutating a single residue inside the pocket could drastically change the “macroscopic” function. To fully understand the exact roles of the protein pocket and chromophore play in governing the biomolecular functionality, insights on the intrinsic molecular timescales (e.g., femtosecond/10-15 s to nanosecond/10-9 s) must be obtained. Ultrafast spectroscopic techniques such as femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations, can illustrate the molecular mechanism upon photoexcitation (photophysics and photochemistry) via ground- and excited-state potential energy surfaces (PESs). The combination of time-resolved electronic and vibrational spectroscopies thus allows the elucidation of intrinsically coupled electronic and nuclear motions during a photoinduced process, and typically provides a non-invasive experimental platform to study biomolecular systems in physiological conditions. Furthermore, the use of light not only allows characterization but also enables potential strategies from photochroism, photodynamic therapy, to light-induced modulation or gain of functionality. First, I investigated a green fluorescent protein (GFP) based calcium biosensor named GEM-GECO1-P377R (P377R for short) in collaboration with the Campbell Lab at the University of Alberta, Canada. Previously, the elucidated excited state proton transfer (ESPT) of the parent biosensor, GEM-GECO1, is crucial in understanding the green and blue fluorescence when calcium ions are free and bound, respectively. This property makes it a powerful emission-ratiometric biosensor. However, mutation of a single proline residue to an arginine changed the prominent blue fluorescence to green upon calcium binding, essentially making it an excitation ratiometric biosensor. In addition, the single-site mutation changed the more hydrophobic environment of the embedded protein chromophore to a hydrophilic and compact environment, promoting a faster ESPT reaction but also creating a trapped excited state population that becomes more photosensitive and photodegradable. One unique spectroscopic finding was the out-of-phase oscillations of the 1265 cm-1 (C–O stretch) and 1575 cm-1 (C=C/C=O stretch) mode intensities in the excited-state FSRS data of the Ca2+-free biosensor following photoexcitation. These oscillations from coherently generated vibrational intensity quantum beats, after Fourier transform analysis, correspond to a low-frequency mode at ~180 cm-1 that intrinsically modulates these higher frequency vibrations via anharmonic coupling, which governs the initial energy transfer between these nuclear motions of the chromophore prior to fluorescence events. Later, I brought my P377R experience of studying the equilibrium dynamics of a calcium-bound versus calcium-free state to dissect the non-equilibrium dynamics of a photoswitchable cyanobacteriochrome (CBCR) called AnPixJg2. This project stemmed from my NSF East Asia and Pacific Summer Institutes for U.S. Graduate Students (EAPSI) and Japan Society for the promotion of Science (JSPS) Fellowship in Summer 2017, and related protein engineering work performed in the Sato Lab at University of Tokyo, Japan. Additional AnPixJg2 sample were provided by the Narikawa Lab at Shizuoka University, Japan. Using fs-TA spectroscopy, we captured the initial reversible photoswitching events between the thermally stable, red-absorbing state (Pr) and the meta-stable, green absorbing conformer (Pg) in real-time. Before converting to the final photoproduct, many photoswitchable proteins like CBCRs generate intermediate Lumi states which are partially-twisted conformers that resemble the photoproduct. The Pr-to-Pg conversion was found to be a more uphill reaction requiring a two-step process (~13 and 217 ps) before reaching an S1/S0 conical intersection (CI). In contrast, the largely downhill Pg-to-Pr conversion requires a much faster (~3 ps) process to reach an S1/S0 CI with a significant rise of the Lumi-G species on the ~30 ps timescale. One of the drawbacks of AnPixJg2 was the incorporation of phycocyanobilin (PCB) as the cofactor. PCB is not biologically available in mammalian cells, which makes it difficult for biomedical applications (e.g., optogenetic for human subjects). Biliverdin (BV) is a known mammalian derivative where the aforementioned Narikawa and Sato Groups incorporated into AnPixJg2 with just four key mutations (AnPixJg2_BV4). Using the field-proven experimental and computational platform that mainly constitutes steady-state and time-resolved electronic and vibrational spectroscopies, aided by density-functional-theory (DFT) calculations, we systematically compared the parent AnPixJg2 with PCB (Apcb) to the mutant AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv). Upon comparison between Apcb and Bpcb, the overall initial photoswitching process remains largely intact, but the mutated pocket allows more room for larger twisting motions to occur faster. The incorporation of BV red-shifts the ground-state absorption bands from Pr/Pg to Pfr/Po due to the extended conjugation of the cofactor. The integrated BV and mutated pocket drastically change the initial photoswitching processes, with both conversions with in either direction (Bbv Po to Pfr and Pfr to Po) requiring a two-step mechanism to reach the CI. Surprisingly, both conversions display similar spectral patterns with almost identical decay constants of ~5 and 35 ps. The major difference was identified to be the relative amplitude weights associated with each temporal component in the fs-TA spectral fitting, which indicates a unique clockwise/counterclockwise reaction pathway (e.g., reversible photoswitching of Bbv inside an engineered CBCR). Notable, I have also contributed to the group research mission and experimental toolset by developing home-built instruments such as a 3D-printed LED box, low- volume flow cell, low-temperature flow cell, and miscellaneous tools to help mix the solution samples in thin quartz cuvettes. Future project investigations include a photodimerizing protein named Vivid and its engineered variants named nMag and pMag. These proteins are flavin adenine dinucleotide (FAD) based proteins with nMag and pMag engineered to have electrostatically charged residues at the interface, which can prevent homodimerization and promote heterodimerization. In addition, these proteins have been implemented with other split proteins like GFP and CRISPR-Cas-9 for specific light activation applications, which could be facilitated and expedited with a molecular mechanistic understanding in real time as shown above by our “Molecular Movie” technology on photosensitive chromophores and cofactors in protein matrix after photons hit.
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  • Intellectual Property (patent, etc.)
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  • 2021-07-17 to 2022-08-03

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