Structural and Functional Studies of Nonribosomal Peptide Synthases

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The world today needs novel solutions to address current challenges in areas spanning areas from sustainable manufacturing to healthcare, and biotechnology offers the potential to help address some of these issues. One tool that offers opportunities across multiple industries is…

The world today needs novel solutions to address current challenges in areas spanning areas from sustainable manufacturing to healthcare, and biotechnology offers the potential to help address some of these issues. One tool that offers opportunities across multiple industries is the use of nonribosomal peptide synthases (NRPSs). These are modular biological factories with individualized subunits that function in concert to create novel peptides.One element at the heart of environmental health debates today is plastics. Biodegradable alternatives for petroleum-based plastics is a necessity. One NRPS, cyanophycin synthetase (CphA), can produce cyanophycin grana protein (CGP), a polymer composed of a poly-aspartic acid backbone with arginine side chains. The aspartic backbone has the potential to replace synthetic polyacrylate, although current production costs are prohibitive. In Chapter 2, a CphA variant from Tatumella morbirosei is characterized, that produces up to 3x more CGP than other known variants, and shows high iCGP specificity in both flask and bioreactor trials. Another CphA variant, this one from Acinetobacter baylyi, underwent rational protein design to create novel mutants. One, G217K, is 34% more productive than the wild type, while G163K produces a CGP with shorter chain lengths. The current structure refined from 4.4Å to 3.5Å. Another exciting application of NRPSs is in healthcare. They can be used to generate novel peptides such as complex antibiotics. A recently discovered iterative polyketide synthase (IPTK), dubbed AlnB, produces an antibiotic called allenomycin. One of the modular subunits, a dehydratase named AlnB_DH, was crystallized to 2.45Å. Several mutations were created in multiple active site residues to help understand the functional mechanism of AlnB_DH. A preliminary holoenzyme AlnB structure at 3.8Å was generated although the large disorganized regions demonstrated an incomplete structure. It was found that chain length is the primary factor in driving dehydratase action within AlnB_DH, which helps lend understanding to this module.
Date Created
2022
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Biocatalytic Halocyclization of Amides Enables By Vanadium Haloperoxidases

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Marine algae are a rich source of bioactive halogenated natural products. Thepresence of these marine natural products has largely been attributed to their biosynthesis by organisms in these environments through a variety of different halogenation mechanisms. One of the key contributors in…

Marine algae are a rich source of bioactive halogenated natural products. Thepresence of these marine natural products has largely been attributed to their biosynthesis by organisms in these environments through a variety of different halogenation mechanisms. One of the key contributors in these halogenation processes are from the vanadium haloperoxidases (VHPOs) class of enzymes. VHPOs perform an electrophilic halogenation through the oxidation of halide ions with hydrogen peroxide as the terminal oxidant. This technique produces an electrophilic halide equivalent that can directly halogenate organic substrates. Despite the numerous known reaction capabilities of this enzyme class, their construction of intramolecular ring formation between a carbon and nitrogen atom has remained unreported. Herein, this study presents a development of a ‘new to nature’ chemical reaction for lactam synthesis. In pursuit of this type of reaction, it was discovered that wild type VHPOs (e.g., Curvularia inaequalis, Corallina officinalis, Corallina pilulifera, Acaryochloria marina) produce halogenated iminolactone compounds from acyclic amides in excellent yields and selectivity greater than 99 percent yield. The extension to chlorocyclizations will also be discussed.
Date Created
2022
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Structure Determination of G Protein Coupled Receptor

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Description
G protein coupled receptors (GPCRs) mediate various of physiologicalactivities which makes them significant drug targets. Determination of atomic level structure of GPCRs facilitates the structure-based drug design. The most widely used method currently for solving GPCR structure is still protein crystallography especially lipidic…

G protein coupled receptors (GPCRs) mediate various of physiologicalactivities which makes them significant drug targets. Determination of atomic level structure of GPCRs facilitates the structure-based drug design. The most widely used method currently for solving GPCR structure is still protein crystallography especially lipidic cubic phase (LCP) crystallization. LCP could mimic the native environment of membrane protein which stable the membrane proteins. Traditional synchrotron source requires large size large size protein crystals (>30 micron) due to the radiation damage during data collection. However, acquiring large sized protein crystals is challenging and not guaranteed practically. In this study, a novel method was developed which combined LCP technology and micro-electron diffraction (MicroED) technology. LCP-MicroED technology was able to collect complete diffraction data sets from serval submicron protein crystals and deliver high resolution protein structures. This technology was first confirmed with soluble protein crystals, proteinase K and small molecule crystals, cholesterol. Furthermore, this novel method was applied to a human GPCR target, Î22- adrenergic receptor (Î22AR). The structure model was successfully built which proved the feasibility of applying LCP-MicroED method to GPCRs and other membrane proteins. Besides, in this research, a novel human GPCR target, human histamine 4 receptor(H4R) was studied. Different constructs were expressed, purified, and characterized. Some key residuals that affect ligand binding were confirmed.
Date Created
2022
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Tungsten Palladium Plates for Assaying H2 Catalysis by Cyt b562-CoPPIX

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Hybrid metalloproteins incorporating synthetic organometallic active sites within a protein scaffold are being researched as viable catalysts for the production of hydrogen fuel. Our group and others have shown that the incorporation of cobalt protoporphyrin IX in cytochrome b₅₆₂ yields…

Hybrid metalloproteins incorporating synthetic organometallic active sites within a protein scaffold are being researched as viable catalysts for the production of hydrogen fuel. Our group and others have shown that the incorporation of cobalt protoporphyrin IX in cytochrome b₅₆₂ yields artificial enzymes that reduce protons to molecular hydrogen in the presence of photoinductive light and photosensitizers. Using random mutagenesis via error-prone PCR we have created a library of mutants to use in directed evolution to optimize hydrogen catalysis, though a challenge in this project is that testing individual variants by gas chromatography is not feasible on a large scale. For this reason, we are developing a gasochromic, hydrogen assay that is based on the interaction of molecular hydrogen with tungsten trioxide with a palladium catalyst. Initially, results show this assay to be qualitatively accurate between trials; however, its application in screening remains a challenge.

Date Created
2022-05
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Syntheses of Non-Canonical Amino Acids Containing Stilbene

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Non-canonical amino acids (NCAAs) can be used in protein chemistry to determine their structures. A common method for imaging proteins is cryo-electron microscopy (cryo-EM) which is ideal for imaging proteins that cannot be obtained in large quantities. Proteins with indistinguishable…

Non-canonical amino acids (NCAAs) can be used in protein chemistry to determine their structures. A common method for imaging proteins is cryo-electron microscopy (cryo-EM) which is ideal for imaging proteins that cannot be obtained in large quantities. Proteins with indistinguishable features are difficult to image using this method due to the large size requirements, therefore antibodies designed specifically for binding these proteins have been utilized to better identify the proteins. By using an existing antibody that binds to stilbene, NCAAs containing this molecule can be used as a linker between proteins and an antibody. Stilbene containing amino acids can be integrated into proteins to make this process more access able. In this paper, synthesis methods for various NCAAs containing stilbene were proposed. The resulting successfully synthesized NCAAs were E)-N6-(5-oxo-5-((4-styrylphenyl) amino) pentanoyl) lysine, (R,E)-2-amino-3-(5-oxo-5-((4-styrylphenyl)amino)pentanamido)propanoic acid, (E)-2-amino-5-(5-oxo-5-((4-styrylphenyl) amino) pentanamido) pentanoic acid. A synthesis for three more shorter amino acids, (R,E)-2-amino-3-(3-oxo-3-((4-styrylphenyl) amino) propanamido) propanoic acid, (E)-2-amino-5-(3-oxo-3-((4-styrylphenyl) amino) propanamido) pentanoic acid, and (E)-N6-(3-oxo-3-((4-styrylphenyl) amino) propanoyl) lysine, is also proposed.

Date Created
2022-05
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Structure Elucidation of Medically Important Proteins by X-ray Crystallography

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Description
Since its conception over a century ago, X-ray crystallography (XRC) has become the most successful method used to elucidate the structures and functions of biological molecules at atomic resolution. The extensive use of XRC has led to meaningful discoveries across…

Since its conception over a century ago, X-ray crystallography (XRC) has become the most successful method used to elucidate the structures and functions of biological molecules at atomic resolution. The extensive use of XRC has led to meaningful discoveries across many scientific fields, notably its contributions to rational drug design. Traditional drug discovery relies on the use of trial-and-error based approaches in cellular and animal models of disease to identify chemical probes that elicit desirable therapeutic effects based off changes in phenotype. However, this approach lacks critical information in regards to the biological target in which the compound interacts with. In contrast, the use of rational drug design presents the opportunity to identify chemical probes that target specific protein targets of known medical importance and study their interactions using three dimensional structures that can be used to suggest new drug candidates. The main focus of my research presented in this dissertation aims to utilize XRC to discover novel therapeutics. In this work, I begin by describing the use of structure-based drug discovery for the rational design of hydrocarbon-stapled peptides that block Focal Adhesion Kinase (FAK) scaffolding in cancer (Chapter 2). FAK is an intracellular tyrosine kinase that has been linked to many cancers through its interaction with Paxillin LD motifs as it relates to tumor growth, invasion, metastasis, and suppression of apoptosis. The results of this study demonstrate the effectiveness hydrocarbon-stapling has on the native Paxillin LD2 sequence with ~50 fold greater binding affinity by surface plasmon resonance (SPR) that can be explained by the unique structural interactions observed by XRC. Next, I present a series of methods which lays the foundations for the discovery of novel anti-bacterial drugs that target 3-Deoxy-D-manno-octulosonate-8-phosphate (KDO8P) Synthase, a critical enzyme in the biosynthesis of gram-negative lipopolysaccharides (Chapter 3).
Date Created
2021
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Hierarchical Assembly of Nucleic Acid/Coiled-Coil Peptide Nanostructures

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Since the inception of DNA nanotechnology, DNA has found itself poised as one of the most robust self-assembling building blocks due to its well understood double helix structure formed by two anti-parallel strands of DNA held together by hydrogen bond…

Since the inception of DNA nanotechnology, DNA has found itself poised as one of the most robust self-assembling building blocks due to its well understood double helix structure formed by two anti-parallel strands of DNA held together by hydrogen bond from nucleobases which also provides the material programmability due to the well-understood Watson Crick base pairing rules. These capabilities have led to the exponential increase in publications showing off intricate and remarkable designs alongside ever-expanding applications. However, as the field expands there is an apparent lack of chemical diversity and functionality. To combat this my research focused on creating hybrid peptide oligonucleotide conjugates (POC) where the conjugated peptide could add chemical and structural diversity using the 20 canonical amino acids and various peptide secondary structures. In this work, I conjugate DNA to the self-assembling peptide building block the coiled coil. The coiled coil motif is formed from the self-assembly of two or more α-helical peptides and, like DNA, the coiled coil has well understood programmability. Together as a conjugate, the DNA and coiled coil, create a new self-assembling building block capable of two orthogonal self-assembling modes that can work in tandem. In this work, I used DNA coiled coil conjugates to show the capability to create first of their kind hybrid DNA/coiled coil one-dimensional fibers (chapter 2), integrate proteins (chapter 3), and to create hybrid cage structures (chapter 4). Finally, a POC hydrogel is created using the polypeptide gelatin with DNA crosslinks to create a reversible stiffening gel using toe-hold mediated strand displacement (chapter 5).
Date Created
2021
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Structural and Functional Studies of Protein-Nanoparticle Complexes and their Interactions

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Description
The list of applications of plasmonic nanoparticles in the fields of energy research, sensing, and diagnostics and therapeutics is continuously growing. Processes for the synthesis of the nanoparticles for such applications should incorporate provision to easily functionalize the particle formed…

The list of applications of plasmonic nanoparticles in the fields of energy research, sensing, and diagnostics and therapeutics is continuously growing. Processes for the synthesis of the nanoparticles for such applications should incorporate provision to easily functionalize the particle formed and should ideally not use toxic reagents or create toxic by-products. The traditional methods of synthesizing nanoparticles generally are energy inefficient, requires stringent conditions such as high temperature, pressure or extreme pH and often produces toxic by-products. Although there exist a few solution-based methods to solve this problem, there is one avenue which has recently gained attention for nanoparticle synthesis: using biomolecules to facilitate nanomaterials synthesis. Using biomolecules for synthesis can provide a template to guide the nucleation process and helps to keep conditions biocompatible while also combining the step of functionalization of the nanoparticle with its synthesis through the biomolecule itself. The dissertation focuses on studying the bio-templated synthesis of two such noble metal nanoparticle which have biomedical applications: gold and platinum. In chapter 2, Gold Nanoparticles (GNP), with long-term stability, were synthesized using Maltose Binding Protein (MBP) as templating agent. The site of gold interaction on MBP was identified by X-ray crystallography. A novel gold binding peptide, AT1 (YPFGGSGGSGM), was designed based on the orientation of the residues in the gold binding site, identified through crystallography. This designed peptide was also shown to have stabilized and affected the growth rate of GNP formation, in similar manner to MBP. Further in chapter 3, a nanosensor was formulated using a variation of this GNP-MBP system, to detect and measure ionizing radiation dose for cancer radiation therapy. Upon exposure to therapeutic levels of ionizing radiation, the MBP‐based sensor system formed gold nanoparticles with a dose‐dependent color that could be used to predict the amount of delivered X‐ray dose. In chapter 4, a similar system of protein templated synthesis was introduced for platinum nanoparticle (PtNP). Here, GroEL, a large homo-tetradecamer chaperone from E.coli, was used as templating and stabilizing agent for reduction of K2PtCl4 ions to form PtNP. To understand how GroEL interacts with the PtNPs and thereby stabilizes them, single-particle cryo-electron microscopy technique was used to model the complex in solution. A 3.8-Å resolution 3D cryo-EM map of GroEL depicting the location of PtNP inside its central cylindrical cavity was obtained. Fitting a GroEL model to the map revealed Arginine-268 from two adjacent subunits of GroEL interacting with the PtNP surface. Finally in chapter 5, a solution to the potential issues of single particle data processing on protein nanoparticle complexes, specifically with 2D classification, was developed by creating masking algorithms.
Date Created
2020
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The Effects of the PsaL Calcium Binding on the Oligomeric and Spectroscopic Properties in Synechocystis sp. PCC 6803

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In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function of these pigment-protein complexes are<br/>conserved between all photoautotrophs, however, the…

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function of these pigment-protein complexes are<br/>conserved between all photoautotrophs, however, the oligomeric structure, as well as the<br/>spectroscopic properties of the PSI complex, differ. In early evolving photoautotrophs, PSI<br/>exists in a trimeric organization, but in later evolving species this was lost and PSI exists solely<br/>as a monomer. While the reasons for a change in oligomerization are not fully understood, one<br/>of the 11 subunits within cyanobacterial PSI, PsaL, is thought to be involved in trimerization<br/>through the coordination of a calcium ion in an adjacent monomer. Recently published<br/>structures have demonstrated that PSI complexes are capable of trimerization without<br/>coordinating the calcium ion within PsaL.<br/>5 Here we explore the role the calcium ion plays in both<br/>the oligomeric and spectroscopic properties in PSI isolated from Synechocystis sp. PCC 6803.

Date Created
2021-05
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Understanding Fluorescent Protein Photoconversion and Assembly of Spinach Rubisco Activase

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Description
Proteins function as molecular machines which perform a diverse set of essential jobs. To use these proteins as tools and manipulate them with directed engineering, a deeper understanding of their function and regulation is needed. In the studies presented here,…

Proteins function as molecular machines which perform a diverse set of essential jobs. To use these proteins as tools and manipulate them with directed engineering, a deeper understanding of their function and regulation is needed. In the studies presented here, the chemical mechanism of a fluorescent protein and the assembly behavior of a chemo-mechanical protein were explored to better understand their operation. In the first study a photoconvertible fluorescent protein (pcFP) was examined which undergoes a photochemical transformation upon irradiation with blue light resulting in an emission wavelength change from green to red. Photo-transformable proteins have been used in high resolution, subcellular biological imaging techniques, and desires to engineer them have prompted investigations into the mechanism of catalysis in pcFPs. Here, a Kinetic Isotope Effect was measured to determine the rate-limiting step of green-to-red photoconversion in the reconstructed Least Evolved Ancestor (LEA) protein. The results provide insight on the process of photoconversion and evidence for the formation of a long-lived intermediate. The second study presented here focuses on the AAA+ protein Rubisco activase (Rca), which plays a critical role in the removal of inhibitors from the carbon-dioxide fixing enzyme Rubisco. Efforts to engineer Rubisco and Rca can be guided by a deeper understanding of their structure and interactions. The structure of higher plant Rca from spinach, and its interactions with its cognate Rubisco, were investigated through negative-stain electron microscopy (EM) and cryo-EM experiments. Multiple types of higher-order oligomers of plant Rca were imaged which have never been structurally characterized, and the AAA+ core of plant Rca was shown to bind Rubisco side-on, similar to bacterial Rca’s. Higher resolution structures of these aggregates and complexes are needed to make definitive observations on protein-protein interactions. However, the results presented here provide evidence for the formation of regulatory structures and an experimental foundation for future exploration of plant Rca through cryo-EM.
Date Created
2020
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