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Scholarly Works (5 results)

  • Thesis
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UC Davis Electronic Theses and Dissertations (2021)

Abstract

Photoreceptors play essential roles in initiating a wide array of biological responses based on external light conditions. Photoreceptors have been shown to be involved in phototropism, shade avoidance, seed germination, vision, and chromic acclimation. These photosensitive proteins initiate these various responses by reversibly photoconverting between a thermally stable dark- adapted state and a meta-stable light-adapted state when a covalently bound chromophore absorbs a photon of light. The chromophore causes the large-scale reorganization of the protein by undergoing a structural or electronic change. Since photoreceptors have evolved to initiate various biological responses as well as improve the efficiency of photosynthesis via chromatic acclimation (converting higher energy light to lower energy light that can be used for photosynthesis), they are also of interest to serve as a basis for designing novel fluorescent probes for medicinal imaging applications or optogenetic tools to selectively initiate and control biological functions in living tissue and study neural circuits at a more fundamental level.

To better understand biological signal transduction and establish the practicality of these photoreceptors for the application outlined above, a molecular level understanding of these photoreceptors is of paramount importance. Key to developing a molecular level understanding of these photoinduced events is the characterization of the photoreceptor forward and reverse reactions with time and temperature dependent spectroscopic techniques. These experiments track changes in the absorption spectra due to the photoreaction, which allows for a mapping of the underlying mechanism and estimations of transient properties (e.g., timescale, energy barriers, quantum efficiencies). Photoreceptors are especially well suited for being studying by time- resolved techniques since the reaction is initiated at a known time point (i.e., photoexcitation). Theanalysis of the resulting time-dependent data sets reveals complex mechanisms, including ground state inhomogeneity, unproductive pathways, and estimation of the quantum efficiencies. Cryokinetic UV-Vis spectroscopy complements the room-temperature primary and secondary transient absorption spectroscopic results as well as isolates quickly evolving intermediates difficult to resolve at room temperature.

In the first part of this dissertation a brief overview to photoswitches, including select molecular photoswitches and biological photoreceptors, is provided and followed by a description of the experimental protocols used in the subsequent chapters. The second part of this dissertation is a collection of studies on the photoinduced dynamics of: i) a synthetic cobalt-dioxolene based photomagnetic molecular photoswitch and ii) several biological photoreceptors including comparative studies on the forward and reverse dynamics of several red/green cyanobacteriochromes (CBCRs) isolated from Nostoc punctiforme, a comparison of the secondary forward and reverse dynamics of the photosensory core of cyanobacterial phytochrome 1 (Cph1∆) isolated from Synechocystis sp. PCC 6803 and the canonical red/green CBCR NpR6012g4 isolated from Nostoc punctiforme, primary forward dynamics of a wild-type and mutated far-red/x CBCR isolated from Anabaena cylindrica PCC 7122, and the photocycle of a novel semisynthetic rhodopsin mimic.

    Cover page: Characterization of the Photodynamics of Photoswitches: A Study of a Photomagnetic Molecular Photoswitch, Phytochromes, Cyanobacteriochromes, and a Cellular Retinoic Acid Binding Protein II Rhodopsin Mimic
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2024)
    The nickel-iron carbon monoxide dehydrogenase (CODH) enzyme uses a heterometallic nickel-iron-sulfur ([NiFe4S4]) cluster to catalyze the reversible interconversion of carbon dioxide (CO2) and carbon monoxide (CO). These reactions are essential for maintaining the global carbon cycle and offer a route towards sustainable greenhouse gas conversion but have not been successfully replicated in synthetic models, in part due to a poor understanding of the natural system. Though the general protein architecture of CODH is known, the electronic structure of the active site is not well-understood, and the mechanism of catalysis remains unresolved. To better understand the CODH enzyme, we have developed a protein-based model containing a heterometallic [NiFe3S4] cluster in the Pyrococcus furiosus (Pf) ferredoxin (Fd). This model binds small molecules such as carbon monoxide and cyanide, analogous to CODH. Multiple redox- and ligand-bound states of [NiFe3S4] Fd (NiFd) have been investigated using a suite of spectroscopic techniques, including resonance Raman, Ni and Fe K-edge X-ray absorption spectroscopy, and electron paramagnetic resonance, to resolve charge and spin delocalization across the cluster, site-specific electron density, and ligand activation. The facile movement of charge through the cluster highlights the fluidity of electron density within iron-sulfur clusters and suggests an electronic basis by which CN- inhibits the native system while the CO-bound state continues to elude isolation in CODH. The detailed characterization of isolable states that are accessible in our CODH model system provides valuable insight into unresolved enzymatic intermediates and offers design principles towards developing functional mimics of CODH.
      Cover page: Electronic isomerism in a heterometallic nickel-iron-sulfur cluster models substrate binding and cyanide inhibition of carbon monoxide dehydrogenase.
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2020)
      The photocycle of a reversible photoisomerizing rhodopsin mimic (M2) is investigated. This system, based on the cellular retinoic acid binding protein, is structurally different from natural rhodopsin systems, but exhibits a similar isomerization upon light irradiation. More specifically, M2 displays a 15-cis to all-trans conversion of retinal protonated Schiff base (rPSB) and all-trans to 15-cis isomerization of unprotonated Schiff base (rUSB). Here we use hybrid quantum mechanics/molecular mechanics (QM/MM) tools coupled with transient absorption and cryokinetic UV-vis spectroscopies to investigate these isomerization processes. The results suggest that primary rPSB photoisomerization of M2 occurs around the C13═C14 double bond within 2 ps following an aborted-bicycle pedal (ABP) isomerization mechanism similar to natural microbial rhodopsins. The rUSB isomerization is much slower and occurs within 48 ps around the C15═N double bond. Our findings reveal the possibility to engineer naturally occurring mechanistic features into artificial rhodopsins and also constitute a step toward understanding the photoisomerization of UV pigments. We conclude by reinforcing the idea that the presence of the retinal chromophore inside a tight protein cavity is not mandatory to exhibit ABP mechanism.
        Cover page: Computational and Spectroscopic Characterization of the Photocycle of an Artificial Rhodopsin
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2021)
        Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward (15ZPr → 15EPg) and reverse (15EPg → 15ZPr) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-Gf intermediate red-shifted from the 15ZPr photoproduct in AnPixJg2. Overall, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φp within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.
          Cover page: Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2015)
          Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts.
            Cover page: Highly evolvable malaria vectors: The genomes of 16 Anopheles mosquitoesCreative Commons 'BY' version 4.0 license
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