Heliobacteria are an anaerobic phototroph that require carbon sources such as pyruvate, <br/>lactate, or acetate for growth (Sattley, et. al. 2008). They are known for having one of the <br/>simplest phototrophic systems, the central component of which is a Type I reaction center (RC) <br/>that pumps protons to generate the electrochemical gradient for making ATP. Heliobacteria <br/>preform cyclic electron flow (CEF) with the RC in the light but can also grow chemotropically in <br/>the dark. Many anaerobes like heliobacteria, such as other members of the class Clostridia, <br/>possess the capability to produce hydrogen via a hydrogenase enzyme in the cell, as protons can <br/>serve as an electron acceptor in anaerobic metabolism. However, the species of heliobacteria <br/>studied here, H. modesticaldum have been seen to produce hydrogen via their nitrogenase <br/>enzyme but not when this enzyme is inactive. This study aimed to investigate if the reason for <br/>their lack of hydrogen production was due to a lack of an active hydrogenase enzyme, possibly <br/>indicating that the genes required for activity were lost by an H. modesticaldum ancestor. This <br/>was done by introducing genes encoding a clostridial [FeFe] hydrogenase from C. thermocellum<br/>via conjugation and measuring hydrogen production in the transformant cells. Transformant cells <br/>produced hydrogen and cells without the genes did not, meaning that the heliobacteria ferredoxin <br/>was capable of donating electrons to the foreign hydrogenase to make hydrogen. Because the <br/>[FeFe] hydrogenase must receive electrons from the cytosolic ferredoxin, it was hypothesized <br/>that hydrogen production in heliobacteria could be used to probe the redox state of the ferredoxin <br/>pool in conditions of varying electron availability. Results of this study showed that hydrogen <br/>production was affected by electron availability variations due to varying pyruvate <br/>concentrations in the media, light vs dark environment, use acetate as a carbon source, and being <br/>provided external electron donors. Hydrogen production, therefore, was predicted to be an <br/>effective indicator of electron availability in the reduced ferredoxin pool.
The use of enzyme-catalyst interfaces is underexplored in the field of biocatalysis, particularly in studies on enabling novel reactivity of enzymes. For this thesis, the HaloTag® protein tagging platform was proposed as a bioconjugation method for a pinacol coupling reaction using lipases, as a model for novel reactivities proceeding via ketyl radical intermediates and hydrogen-bonding-facilitated redox attenuation. After an initial lipase screening of 9 lipases, one lipase (Candida rugosa) was found to perform the pinacol coupling of p-anisaldehyde under standard conditions (fluorescein and 530nm light, 3% yield). Based on a retrosynthetic analysis for the photocatalyst-incorporated HaloTag® linker, the intermediates haloamine 1 and aldehyde 6 were synthesized. Further experiments are underway or planned to complete linker synthesis and conduct pinacol coupling experiments with a bioconjugated system. This project underscores the promising biocatalytic promiscuity of lipases for performing reactions proceeding through ketyl radical intermediates, as well as the underdeveloped potential of incorporating bioengineering principles like bioconjugation into biocatalysis to overcome kinetic barriers to electron transfer and optimize biocatalytic reactions.
DNA nanotechnology, the self-assembly of DNA into 2D and 3D nanoscale structures facilitated via Watson and Crick base pairing, provides alternative solutions for biomedical challenges, especially for therapeutic cargo delivery, because it is easily fabricated, exhibits low cytotoxicity, and high biocompatibility. However, the stability of these DNA nanostructures (DN) under cellular environment presents an issue due to their requirements for high salt conditions and susceptibility to nuclease degradation. Furthermore, DNs are typically trapped in endolysosomal compartments rather than the cytosol, where most of their cargo must be delivered. Many attempts to mitigate the stability issue have been made in recent years. Previously, our lab designed an endosomal escape peptide, Aurein 1.2 (denoted “EE, for endosomal escape)”, combined with a decalysine sequence (K10) proven to electrostatically adhere to and protect DNs under cell culture conditions. Unfortunately, this molecule, termed K10-EE, only resulted in endosomal escape in absence of serum due to formation of a protein corona on the surface of the coated DN.6 Therefore, we now propose to electrostatically coat the DN with a polymer composed of decalysine (K10), polyethylene glycol (PEG, which demonstrates antibiofouling properties), and peptide EE: K10- PEG1k-EE. Described herein are the attempted synthetic schemes of K10-PEG1k-EE, the successful synthesis of alternative products, K10-(EK)5 and K10-(PEG12)2-EE, and their resulting impacts on DN stability under biological conditions. Coating of the K10-(EK)5 with a DNA barrel origami demonstrated inefficient stabilizing capability in serum. Future studies include testing K10- (PEG12)2-EE protection for a variety of nucleic acid-based structures.
This qualitative study sought to investigate the potential reaction between the 3,3',5,5'-tetramethylbenzidine (TMB) radical and LAF-1 RGG, the N-terminus domain of an RNA helicase which functions as a coacervating intrinsically disordered protein. The study was performed by adding horseradish peroxidase to a solution containing TMB and either LAF-1 or tyrosine in various concentrations, and monitoring the output through UV-Vis spectroscopy. The reacted species was also analyzed via MALDI-TOF mass spectrometry. UV-Vis spectroscopic monitoring showed that in the presence of LAF-1 or tyrosine, the reaction between HRP and TMB occurred more quickly than the control, as well as in the highest concentration of LAF-1, the evolution of a peak at 482 nm. The analysis through MALDI-TOF spectrometry showed the development of a second peak likely due to the reaction between LAF-1 and TMB, as the Δ between the peaks is 229 Da and the size of the TMB species is 240 Da.
sustainable, carbon neutral energy source to curb the reliance on fossil fuel combustion as the primary source of energy. To overcome this challenge, research efforts have turned to biology for inspiration, as nature is adept at inter-converting low molecular weight precursors into complex molecules. A number of inorganic catalysts have been reported that mimic the active sites of energy-relevant enzymes such as hydrogenases and carbon monoxide dehydrogenase. However, these inorganic models fail to achieve the high activity of the enzymes, which function in aqueous systems, as they lack the critical secondary-shell interactions that enable the active site of enzymes to outperform their organometallic counterparts.
To address these challenges, my work utilizes bio-hybrid systems in which artificial proteins are used to modulate the properties of organometallic catalysts. This approach couples the diversity of organometallic function with the robust nature of protein biochemistry, aiming to utilize the protein scaffold to not only enhance rates of reaction, but also to control catalytic cycles and reaction outcomes. To this end, I have used chemical biology techniques to modify natural protein structures and augment the H2 producing ability of a cobalt-catalyst by a factor of five through simple mutagenesis. Concurrently I have designed and characterized a de novo peptide that incorporates various iron sulfur clusters at discrete distances from one another, facilitating electron transfer between the two. Finally, using computational methodologies I have engineered proteins to alter the specificity of a CO2 reduction reaction. The proteins systems developed herein allow for study of protein secondary-shell interactions during catalysis, and enable structure-function relationships to be built. The complete system will be interfaced with a solar fuel cell, accepting electrons from a photosensitized dye and storing energy in chemical bonds, such as H2 or methanol.
Additionally, two different approaches to incorporate non-natural organometallic catalysts into protein matrix are discussed. First, cobalt protoporphyrin IX was incorporated into cytochrome b562 to produce a water-soluble proton and CO2 reduction catalyst that is active upon irradiation in the presence of a photosensitizer. The effect of the porphyrin axial ligands provided by the protein environment has been investigated by introducing mutations into the native scaffold, indicating that catalytic activity of proton reduction is dependent on axial coordination to the porphyrin. It is also shown that effects of the protein environment are not directly transferred when applied to other reactions, such as CO2 reduction.
Inspired by the active site of [FeFe]-hydrogenases, the second approach is based on the stereoselective preparation of a novel amino acid bearing a 1,2-benzenedithiol side chain. This moiety can serve as an anchoring point for the introduction of metal complexes into protein matrices. By doing so, this strategy enables the study of protein interactions with non-natural cofactors and the effects that it may have on catalysis. The work developed herein lays a foundation for furthering the study of the use of proteins as suitable environments for tuning the activity of organometallic catalysts in aqueous conditions, and interfacing these systems with other supporting units into supramolecular assemblies.
A unique computational approach combined with in vitro telomerase activity reconstitution studies was used to identify 83 novel TRs from 10 animal kingdom phyla spanning 18 diverse classes from the most basal sponges to the late evolving vertebrates. This revealed that three structural domains, pseudoknot, a distal stem-loop moiety and box H/ACA, are conserved within TRs from basal groups to vertebrates, while group-specific elements emerge or disappear during animal TR evolution along different lineages.
Next the corn-smut fungus Ustilago maydis TR was identified using an RNA-immunoprecipitation and next-generation sequencing approach followed by computational identification of TRs from 19 additional class Ustilaginomycetes fungi, leveraging conserved gene synteny among TR genes. Phylogenetic comparative analysis, in vitro telomerase activity and TR mutagenesis studies reveal a secondary structure of TRs from higher fungi, which is also conserved with vertebrates and filamentous fungi, providing a crucial link in TR evolution within the opisthokonta super-kingdom.
Lastly, work by collabarotors from Texas A&M university and others identified the first bona fide TR from the model plant Arabidopsis thaliana. Computational analysis was performed to identify 85 novel AtTR orthologs from three major plant clades: angiosperms, gymnosperms and lycophytes, which facilitated phylogenetic comparative analysis to infer the first plant TR secondary structural model. This model was confirmed using site-specific mutagenesis and telomerase activity assays of in vitro reconstituted enzyme. The structures of plant TRs are conserved across land plants providing an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.