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- All Subjects: Biochemistry
Description
G protein-coupled receptors, or GPCRs, are receptors located within the membrane of cells that elicit a wide array of cellular responses through their interactions with G proteins. Recent advances in the use of lipid cubic phase (LCP) for the crystallization of GPCRs, as well as increased knowledge of techniques to improve receptor stability, have led to a large increase in the number of available GPCR structures, despite historic difficulties. This project is focused on the histamine family of receptors, which are Class A GPCRs that are involved in the body’s allergic and inflammatory responses. In particular, the goal of this project was to design, express, and purify histamine receptors with the ultimate goal of crystallization. Successive rounds of optimization included the use of recombinant DNA techniques in E.coli to truncate sections of the proteins and the insertion of several fusion partner proteins to improve receptor expression and stability. All constructs were expressed in a Bac-to-Bac baculovirus expression system using Sf9 insect cells, solubilized using n-Dodecyl-β-D-Maltoside (DDM), and purified using immobilized metal affinity chromatography. Constructs were then analyzed by SDS-Page, Western blot, and size-exclusion chromatography to determine their presence, purity, and homogeneity. Along with their expression data from insect cells, the most stable and homogeneous construct from each round was used to design successive optimizations. After 3 rounds of construct design for each receptor, much work remains to produce a stable sample that has the potential to crystallize. Future work includes further optimization of the insertion site of the fusion proteins, ligand screening for co-crystallization, optimization of purification conditions, and screening of potential thermostabilizing point mutations. Success in solving a structure will allow for a more detailed understanding of the receptor function in addition to its vital use in rational drug discovery.
ContributorsCosgrove, Steven Andrew (Author) / Liu, Wei (Thesis director) / Mills, Jeremy (Committee member) / Mazor, Yuval (Committee member) / W. P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The understanding of protein functions in vivo is very important since the protein is the building block of a cell. Cryogenic electron microscopy (cryo-EM) is capable of visualizing protein samples in their near-native states in high-resolution details. Cryo-EM enables the visualization of biomolecular structures at multiscale ranging from a cellular structure to an atomic structure of protein subunit.Neurodegenerative diseases, like Alzheimer’s disease and frontotemporal dementia, have multiple dysregulated signaling pathways. In my doctoral studies, I investigated two protein complexes relevant to these disorders: one is the proNGF- p75 neurotrophin receptor (p75NTR)- sortilin neurotrophin complex and the other is the p97R155H mutant complex. The neurotrophins are a family of soluble basic growth factors involved in the development, maintenance, and proliferation of neurons in the central nervous system (CNS) and peripheral nervous system (PNS). The ligand for the neuronal receptors dictates the fate of the neuronal cells. My studies focused on understanding the binding interfaces between the proteins in the proNGF-p75NTR-sortilin neuronal apoptotic complex. I have performed the biochemical characterization of the complex to understand how the complex formation occurs.
Single amino-acid mutation of R155H on the N-domain of p97 is known to be the prevalent mutation in 40% patients suffering from neurodegenerative disease. The p97R155H mutant exhibits abnormal ATPase activity and cofactor dysregulation. I pursued biochemical characterization in combination with single-particle cryo-EM to explore the interaction of p97R155H mutant with its cofactor p47 and determined the full-length structures of the p97R155H-p47 assemblies for the first time. About 40% p97R155H organizes into higher order dodecamers, which lacks nucleotide binding, does not bind to p47, and closely resembles the structure of p97 bound with an adenosine triphosphate (ATP)-competitive inhibitor, CB-5083, suggesting an inactive state of the p97R155H mutant. The structures also revealed conformational changes of the arginine fingers which might contribute to the elevated p97R155H ATPase activity. Because the D1-D2 domain communication is important in regulating the ATPase function, I further studied the functions of the conserved L464 residue on the D1-D2 linker using mutagenesis and single-particle cryo-EM. The biochemical and structural results suggested the torsional constraint of the D1-D2 linker likely modulates the D2 ATPase activity. Our studies thus contributed to develop deeper knowledge of the intricate cellular mechanisms and the proteins affected in disease pathways.
ContributorsNandi, Purbasha (Author) / Chiu, Po-Lin (Thesis advisor) / Mazor, Yuval (Committee member) / Hansen, Debra T (Committee member) / Arizona State University (Publisher)
Created2022
Description
First evolving in cyanobacteria, the light reactions of oxygenic photosynthesis are carried out by the membrane proteins, photosystem II and photosystem I, located in the thylakoid membrane. Both utilize light captured by their core antenna systems to catalyze a charge separation event at their respective reaction centers and energizes electrons to be transferred energetically uphill, eventually to be stored as a high energy chemical bond. These protein complexes are highly conserved throughout different photosynthetic lineages and understanding the variations across species is vital for a complete understanding of how photosynthetic organisms can adapt to vastly different environmental conditions. Most knowledge about photosynthesis comes from only a handful of model organisms grown under laboratory conditions. Studying model organisms has facilitated major breakthroughs in understanding photosynthesis, however, due to the vast global diversity of environments where photosynthetic organisms are found, certain aspects of this process may be overlooked or missed by focusing on a select group of organisms optimized for studying in laboratory conditions. This dissertation describes the isolation of a new extremophile cyanobacteria, Cyanobacterium aponinum 0216, from the Arizona Sonoran Desert and its innate ability to grow in light intensities that exceed other model organisms. A structure guided approach was taken to investigate how the structure of photosystem I can influence the spectroscopic properties of chlorophylls, with a particular focus on long wavelength chlorophylls, in an attempt to uncover if photosystem I is responsible for high light tolerance in Cyanobacterium aponinum 0216. To accomplish this, the structure of photosystem I was solved by cryogenic electron microscopy to 2.7-anstrom resolution. By comparing the structure and protein sequences of Cyanobacterium aponinum to other model organisms, specific variations were identified and explored by constructing chimeric PSIs in the model organism Synechocystis sp. PCC 6803 to determine the effects that each specific variation causes. The results of this dissertation describe how the protein structure and composition affect the spectroscopic properties of chlorophyll molecules and the oligomeric structure of photosystem I, possibly providing an evolutionary advantage in the high light conditions observed in the Arizona Sonoran Desert.
ContributorsDobson, Zachary (Author) / Fromme, Petra (Thesis advisor) / Mazor, Yuval (Thesis advisor) / Redding, Kevin (Committee member) / Moore, Gary (Committee member) / Arizona State University (Publisher)
Created2022
Description
The growing global energy demand coupled with the need for a low-carbon economy requires innovative solutions. Microalgal oxygenic photosynthesis provides a sustainable platform for efficient capture of sunlight and storage of some of the energy in the form of reduced carbon derivatives. Under certain conditions, the photosynthetic reductant can be shunted to molecular hydrogen production, yet the efficiency and longevity of such processes are insufficient. In this work, re-engineering of the heterodimeric type I reaction center, also known as photosystem I (PSI), in the green microalga Chlamydomonas reinhardtii was shown to dramatically change algal metabolism and improve photobiological hydrogen production in vivo. First, an internal fusion of the small PsaC subunit of PSI harboring the terminal photosynthetic electron transport chain cofactors with the endogenous algal hydrogenase 2 (HydA2) was demonstrated to assemble on the PSI core in vivo, albeit at ~15% the level of normal PSI accumulation, and make molecular hydrogen from water oxidation. Second, the more physiologically active algal endogenous hydrogenase 1 (HydA1) was fused to PsaC in a similar fashion, resulting in improved levels of accumulation (~75%). Both algal hydrogenases chimeras remained extremely oxygen sensitive and benefited from oxygen removal methods. On the example of PSI-HydA1 chimera, it was demonstrated that the active site of hydrogenase can be reactivated in vivo after complete inactivation by oxygen without the need for new polypeptide synthesis. Third, the hydrogenase domain of Megasphaera elsdenii bacterial hydrogenase (MeHydA) was also fused with psaC, resulting in expression of a PSI-hydrogenase chimera at ~25% the normal level. The heterologous hydrogenase chimera could be activated with the algal maturation system, despite only 32 % sequence identity (43 % similarity). All constructs demonstrated diminished ability to reduce PSI electron acceptors (ferredoxin and flavodoxin) in vitro and indirect evidence indicated that this was true in vivo as well. Finally, chimeric design considerations are discussed in light of the models generated by Alphafold2 and how could they be used to further optimize stability of the PSI-hydrogenase chimeric complexes.
ContributorsKanygin, Andrey (Author) / Redding, Kevin E (Thesis advisor) / Jones, Anne K (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2022
Description
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.
ContributorsDavis, Morgan (Author) / Ghirlanda, Giovanna (Thesis director) / Heyden, Matthias (Committee member) / Mazor, Yuval (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor)
Created2022-12
Description
The objective of this study is to create a spectrophotometric assay that can measure quinone reduction in the HbRC. The key techniques used in the project consisted of a PCR, a pseudo golden gate, a transformation into E. coli, a conjugation into Heliomicrobium modesticaldum, a growth study, a HbRC prep, and absorbance spectroscopy. PCR was crucial for amplifying the Cyt c553-PshX gene for the pseudo golden gate. The pseudo golden gate ligated Cyt c553-PshX into the plasmid pMTL86251 in order to transform the plasmid with the desired gene into the E. coli strain S17-1. This E. coli strain allows for conjugation into H. modesticaldum. H. modesticaldum cannot uptake DNA by itself, so the E. coli creates a pilus to transfer the desired plasmid to H. modesticaldum. The growth study was crucial for determining if H. modesitcaldum could be induced using xylose without killing the cells or inhibiting the growth in such a way that the project could not be continued. The HbRC prep was used to isolate and purify the Cyt c553-PshX protein. Absorbance spectroscopy and JTS kinetic assay was used to characterize and confirm that the protein eluted from the affinity column was Cyt c553-PshX. The results of the absorbance spectra and JTS kinetic assay confirmed that Cyt c553-PshX was not made. The study is currently being continued using a new system that utilizes SpyCatcher SpyTag covalent linkages in order to attach cytochrome to reduce P800 to the HbRC.
ContributorsBarnes, Katherine (Author) / Redding, Kevin (Thesis director) / Mazor, Yuval (Committee member) / Singharoy, Abhishek (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / Department of English (Contributor)
Created2022-12
Description
Particulate Guanylyl Cyclase Receptor A (pGC-A) is an atrial natriuretic peptide receptor, which plays a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain of pGC-A interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop a method that can regulate pGC-A, structural information regarding its intact form is necessary. Currently, only the extracellular domain structure of rat pGC-A has been determined. However, structural data regarding the transmembrane domain, as well as functional intracellular domain regions, need to be elucidated.This dissertation presents detailed information regarding pGC-A expression and optimization in the baculovirus expression vector system, along with the first purification method for purifying functional intact human pGC-A. The first in vitro evidence of a purified intact human pGC-A tetramer was detected in detergent micellar solution. Intact pGC-A is currently proposed to function as a homodimer. Upon analyzing my findings and acknowledging that dimer formation is required for pGC-A functionality, I proposed the first tetramer complex model composed of two functional subunits (homodimer). Forming tetramer complexes on the cell membrane increases pGC-A binding efficiency and ligand sensitivity.
Currently, a two-step mechanism has been proposed for ATP-dependent pGC-A signal transduction. Based on cGMP functional assay results, it can be suggested that the binding ligand also moderately activates pGC-A, and that ATP is not crucial for the activation of guanylyl cyclase. Instead, three modulators can regulate different activation levels in intact pGC-A.
Crystallization of purified intact pGC-A was performed to determine its structure. During the crystallization condition screening process, I successfully selected seven promising initial crystallization conditions for intact human pGC-A crystallization. One selected condition led to the formation of excellent needle-shaped crystals. During the serial crystallography diffraction experiment, five diffraction patterns were detected. The highest diffraction resolution spot reached 3 Å.
This work will allow the determination of the intact human pGC-A structure while also providing structural information on the protein signal transduction mechanism. Further structural knowledge may potentially lead to improved drug design. More precise mutation experiments could help verify the current pGC-A signal transduction and activation mechanism.
ContributorsZhang, Shangji (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
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 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.
ContributorsJing, Liang (Author) / Mazor, Yuval (Thesis advisor) / Mills, Jeremy (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2022
Description
Cryogenic Electron Microscopy (Cryo-EM) is a method that can be used for studying the structure of biological systems. Biological samples are frozen to cryogenic temperatures and embedded in a vitreous ice when they are imaged by electrons. Due to its ability to preserve biological specimens in near-native conditions, cryo-EM has a significant contribution to the field of structural biology.Single-particle cryo-EM technique was utilized to investigate the dynamical characteristics of various protein complexes such as the Nogo receptor complex, polymerase ζ (Polζ) in yeast and human integrin ⍺vβ8-pro-TGFβ1-GARP complex. Furthermore, I proposed a new method that can potentially improve the sample preparation for cryo-EM. The Nogo receptor complex was expressed using baculovirus expression system in sf9 insect cells and isolated for structural studies. Nogo receptor complex was found to have various stoichiometries and interactions between individual proteins. A structural investigation of the yeast apo polymerase ζ holoenzyme was also carried out. The apo Polζ displays a concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. Furthermore, a lysine residue that obstructs the DNA-binding channel in apo Polζ was found and suggested a gating mechanism. In addition, cryo-EM studies of the human integrin ⍺vβ8-pro-TGFβ1-GARP complex was conducted to assess its dynamic interactions. The 2D classifications showed the ⍺vβ8-pro-TGFβ1-GARP complex is highly flexible and required several sample preparation techniques such as crosslinking and graphene oxide coating to improve protein homogeneity on the EM grid. To overcome challenges within the cryo-EM technique such as particle adsorption on air-water interface, I have documented a collaborative work on the development and application of lipid monolayer sandwich on cryo-EM grid. Cryogenic electron tomography (cryo-ET) along with cryo-EM were used to study the characteristics of lipid monolayer sandwich as a potential protective layer for EM grid. The cryo-ET results demonstrated that the thickness of lipid monolayer is adequate for single-particle cryo-EM processing. Furthermore, there was no appearance of preferred orientations in cryo-EM and cryo-ET images. To establish that this method is actually beneficial, more data must be collected, and high-resolution structures of protein samples must be obtained using this methodology.
ContributorsTruong, Chloe Du (Author) / Chiu, Po-Lin (Thesis advisor) / Liu, Wei (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
Description
In algae, the Mutant Affecting Retrograde Signaling (MARS1) Kinase plays a critical role in the chloroplast unfolded protein response (cpUPR) when the chloroplast faces proteotoxic stress4. The MARS1 protein is relatively unknown in terms of structure and function. However, there has been ample research performed on the main pathway associated with the MARS1 protein, the cpUPR. The exact mechanism of why MARS1 is necessary for the cpUPR is still unknown. Our structural and biochemical studies will help develop a better understanding of the MARS1 structure, and the role it plays in the cpUPR. The MARS1 expression construct will be assembled following the yeast golden gate (yGG) assembly protocol. Here, we will attempt to recombinantly express MARS1 kinase in Saccharomyces cerevisiae to provide insights into the protein.
ContributorsHeeres, Nicholas (Author) / Mazor, Yuval (Thesis director) / Chiu, Po Lin (Committee member) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05