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

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

Watching an Engineered Calcium Biosensor Glow: Altered Reaction Pathways before Emission

Biosensors have become an indispensable tool set in life sciences. Among them, fluorescent protein-based biosensors have great biocompatibility and tunable emission properties but their development is largely on trial and error. To facilitate a rational design, we implement tunable femtosecond stimulated Raman spectroscopy, aided by transient absorption and quantum calculations, to elucidate the working mechanisms of a single-site Pro377Arg mutant of an emission ratiometric Ca2+ biosensor based on a green fluorescent protein-calmodulin complex. Comparisons with the parent protein and the Ca2+-free/bound states unveil more structural inhomogeneity yet an overall faster excited-state proton-transfer (ESPT) reaction inside the Ca2+-bound biosensor. The correlated photoreactant and photoproduct vibrational modes in the excited state reveal more chromophore twisting and trapping in the Ca2+-bound state during ESPT and the largely conserved chromophore dynamics in the Ca2+-free state from parent protein. The uncovered structural dynamics insights throughout an ESPT reaction inside a calcium biosensor provide important design principles in maintaining a hydrophilic, less compact, and more homogeneous environment with directional H-bonding (from the chromophore to surrounding protein residues) via bioengineering methods to improve the ESPT efficiency and quantum yield while maintaining photostability

Molecular Characterization of the Tumor Suppressor Candidate 5 Gene: Regulation by PPARγ and Identification of TUSC5 Coding Variants in Lean and Obese Humans

Tumor suppressor candidate 5 (TUSC5) is a gene expressed abundantly in white adipose tissue (WAT), brown adipose tissue (BAT), and peripheral afferent neurons. Strong adipocyte expression and increased expression following peroxisome proliferator activated receptor γ (PPARγ) agonist treatment of 3T3-L1 adipocytes suggested a role for Tusc5 in fat cell proliferation and/or metabolism. However, the regulation of Tusc5 in WAT and its potential association with obesity phenotypes remain unclear. We tested the hypothesis that the TUSC5 gene is a bona fide PPARγ target and evaluated whether its WAT expression or single-nucleotide polymorphisms (SNPs) in the TUSC5 coding region are associated with human obesity. Induction of Tusc5 mRNA levels in 3T3-L1 adipocytes by troglitazone and GW1929 followed a dose-response consistent with these agents' binding affinities for PPARγ. Chromatin immunoprecipitation (ChIP) experiments confirmed that PPARγ protein binds a ∼ −1.1 kb promotor sequence of murine TUSC5 transiently during 3T3-L1 adipogenesis, concurrent with histone H3 acetylation. No change in Tusc5 mRNA or protein levels was evident in type 2 diabetic patients treated with pioglitazone. Tusc5 expression was not induced appreciably in liver preparations overexpressing PPARs, suggesting that tissue-specific factors regulate PPARγ responsiveness of the TUSC5 gene. Finally, we observed no differences in Tusc5 WAT expression or prevalence of coding region SNPs in lean versus obese human subjects. These studies firmly establish the murine TUSC5 gene locus as a PPARγ target, but the significance of Tusc5 in obesity phenotypes or in the pharmacologic actions of PPARγ agonists in humans remains equivocal

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm−1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances