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Description
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
ContributorsLi, Dianfan (Author) / Stansfeld, Phillip J. (Author) / Sansom, Mark S. P. (Author) / Keogh, Aaron (Author) / Vogeley, Lutz (Author) / Howe, Nicole (Author) / Lyons, Joseph A. (Author) / Aragao, David (Author) / Fromme, Petra (Author) / Fromme, Raimund (Author) / Basu, Shibom (Author) / Grotjohann, Ingo (Author) / Kupitz, Christopher (Author) / Rendek, Kimberley (Author) / Weierstall, Uwe (Author) / Zatsepin, Nadia (Author) / Cherezov, Vadim (Author) / Liu, Wei (Author) / Bandaru, Sateesh (Author) / English, Niall J. (Author) / Gati, Cornelius (Author) / Barty, Anton (Author) / Yefanov, Oleksandr (Author) / Chapman, Henry N. (Author) / Diederichs, Kay (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Seibert, M. Marvin (Author) / Caffrey, Martin (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2015-12-17
Description
Non-alcoholic fatty liver disease occurs when triglycerides are stored in the liver leading to irreversible scarring and damage of liver tissue. Inside the liver, adipose triglyceride lipase is responsible for the breaking down of triglycerides and is regulated by the inhibitor g0/g1 switch gene 2 (G0S2). G0S2 is proposed to be one of the targets against drug design for non-alcoholic fatty liver disease, and more information is needed on the structure of this protein to aid in drug discovery. Here I describe the expression of G0S2 in an E. coli system as well as purification and biophysical characterization of a functional G0S2 in amounts viable for solution state Nuclear Magnetic Resonance (NMR) spectroscopy. Initial spectra of the isotopically labeled protein show well dispersed 15N resonance lines, clean 13C resonances, and dominant a-helices characteristics. These results show that a prepared G0S2 construct is suitable for solution NMR such that 20 amino acids are now assigned in the G0S2 portion of the protein, allowing for further NMR work with this protein for structural discovery. Further work with a large oligomeric complex of G0S2 with Maltose Binding Protein also shows promise for future cryo-EM work.
ContributorsMoran, Michael William (Author) / Fromma, Petra (Thesis advisor) / Guo, Jia (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
Description
G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
ContributorsGeiger, James (Author) / Liu, Wei (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
Description
This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics.
Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.
Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.
Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
ContributorsNagaratnam, Nirupa (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Thesis advisor) / Van Horn, Wade (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
Description
This work comprises a cumulative effort to provide analysis of proteins relevant to understanding and treating human disease. This dissertation focuses on two main protein complexes: the structure of the Chimp adenovirus Y25 capsid assembly, as used in the SARS-CoV-2 vaccine, Vaxzveria, and the Dbl family RhoGEF (guanosine exchange factor) Syx and its associated small G protein, RhoA. The course of research was influenced heavily by the onset of the Covid-19 pandemic and associated lockdown, which pushed anyone with the means to do meaningful research to shift priorities towards addressing the greatest public health crisis since the 1918 flu pandemic.
Analysis of the Syx-RhoA complex for the purposes of structurally guided drug design was initially the focus of heavy optimization efforts to overcome the numerous challenges associated with expression, purification, and handling of this protein. By analyzing E. Coli derived protein new important knowledge was gained about this protein’s biophysical characteristics which contribute to its behavior and may inform drug design efforts. Expression in SF9 insect cells resulted in promising conditions for production of homogeneous and monodispersed protein. Homology modeling and molecular dynamics simulation of this protein support hypotheses about its interactions with both RhoA as well as regions of the cytoplasmic leaflet of the cell membrane.
Structural characterization of ChAdOx1, the adenoviral vector used in the AstraZeneca Covid-19 vaccine, Vaxzveria resulted in the highest resolution adenovirus structure ever solved (3.07Å). Subsequent biochemical analysis and computational simulations of PF4 with the ChAdOx1 capsid reveal interactions with important implications for vaccine induced thrombocytic throbocytopenia syndrome, a disorder observed in approximately 0.000024% of patients who receive Vaxzveria.
ContributorsBoyd, Ryan J (Author) / Fromme, Petra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2021
Description
Structural-based drug discovery is becoming the essential tool for drug development withlower cost and higher efficiency compared to the conventional method. Knowledge of the
three-dimensional structure of protein targets has the potential to accelerate the process
for screening drug candidates. X-ray crystallography has proven to be the most used and
indispensable technology in structural-based drug discovery. The provided
comprehensive structural information about the interaction between the disease-related
protein target and ligand can guide the chemical modification on the ligand to improve
potency and selectivity. X-ray crystallography has been upgraded from traditional
synchrotron to the third generation, which enabled the surge of the structural
determination of macromolecular. The introduction of X-ray free electron laser further
alleviated the uncertain and time-consuming crystal size optimization process and
extenuated the radiation damage by “diffraction before destruction”. EV-D68 2A
protease was proved to be an important pharmaceutical target for acute flaccid myelitis.
This thesis reports the first atomic structure of the EV-D68 2A protease and the structuresof its two mutants, revealing it adopting N-terminal four-stranded sheets and C-terminal
six-stranded ß-barrels structure, with a tightly bound zinc atom. These structures will
guide the chemical modification on its inhibitor, Telaprevir. Integrin ⍺Mβ2 is an integrin
with the α I-domain, related to many immunological functions including cell
extravasation, phagocytosis, and immune synapse formation, so studying the molecular
ligand-binding mechanism and activation mechanism of ⍺Mβ2 is of importance. This
thesis uncovers the preliminary crystallization condition of ⍺Mβ2-I domain in complex
with its ligand Pleiotrophin and the initial structural model. The structural model shows consistency with the previous hypothesis that the primary binding sites are metal iondependent
adhesion sites on ⍺Mβ2-I domain and the thrombospondin type-1 repeat (TSR)
domains of Pleiotrophin. Drug molecules with high potency and selectivity can be
designed based on the reported structures of the EV-D68 2A protease and ⍺Mβ2-I domain
in the future.
ContributorsLiu, Chang (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
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
Serial femtosecond crystallography (SFX) uses diffraction patterns from crystals delivered in a serial fashion to an X-Ray Free Electron Laser (XFEL) for structure determination. Typically, each diffraction pattern is a snapshot from a different crystal. SFX limits the effect of radiation damage and enables the use of nano/micro crystals for structure determination. However, analysis of SFX data is challenging since each snapshot is processed individually.
Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis.
A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools.
Many photosystem II (PSII) dataset have been collected at XFELs, several of which are time-resolved (containing both dark and laser illuminated frames). Comparison of light and dark datasets requires understanding systematic errors that can be introduced during data analysis. This dissertation describes data analysis of PSII datasets with a focus on the effect of parameters on later results. The influence of the subset of data used in the analysis is also examined and several criteria are screened for their utility in creating better subsets of data. Subsets are compared with Bragg data analysis and continuous diffuse scattering data analysis.
A new tool, DatView aids in the creation of subsets and visualization of statistics. DatView was developed to improve the loading speed to visualize statistics of large SFX datasets and simplify the creation of subsets based on the statistics. It combines the functionality of several existing visualization tools into a single interface, improving the exploratory power of the tool. In addition, it has comparison features that allow a pattern-by-pattern analysis of the effect of processing parameters. \emph{DatView} improves the efficiency of SFX data analysis by reducing loading time and providing novel visualization tools.
ContributorsStander, Natasha (Author) / Fromme, Petra (Thesis advisor) / Zatsepin, Nadia (Thesis advisor) / Kirian, Richard (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2019
Description
G protein-coupled receptors (GPCRs) are a large family of proteins involved in the cell signaling and regulation of many biological and pathological processes in the human body. To fully understand their functions, various approaches are needed. This work combines several techniques to advance the study of GPCRs with the overarching goal of pursuing X-ray crystallization using lipidic cubic phase (LCP). In meso, or LCP crystallization method involves imbedding the GPCR into a lipid membrane-mimetic material which spontaneously forms when monoacylglycerols (MAGs) are mixed at the correct hydration level and temperature. It provides a stable environment for GPCRs and has been established as the most common method to resolve structural details of GPCRs (Chapter 2). Yet, before crystallization, GPCRs need to be put through several rounds of optimization of the construct design, including truncation of N- and C- termini, fusing different soluble proteins, and mutating the receptor (Chapter 3). Other methods were also used to gain structural insights into GPCR interactions, such as coarse-grained molecular dynamic simulations, which showed the specific regions of interactions with cholesterol molecules imbedded in the membranes (Chapter 4). This study demonstrated β2-adrenergic receptor (β2AR), a GPCR, as a model of a cholesterol-sensitive receptor. Mutations were made to test the effect of removing specific residues of interest on cholesterol stabilization through the LCP-Tm assay, producing results that align with the simulation data. Finally, the goal of the last study is to provide a guide to identify which host lipids form stable LCP phases for different applications (Chapter 5). Small angle X-ray scattering is used to identify phases in hundreds of different precipitant conditions in the search of suitable host lipid for LCP studies. The results present a systematic overview of the compatibility of common MAGs by screening them against different precipitant solutions including varying salts and polyethylene glycol (PEG) concentrations, different PEG sizes, the presence of detergent or protein in the sample, and the addition of cholesterol. Together, these studies present a variety of methods to advance the structural studies of GPCRs using LCP
ContributorsAL-SAHOURI, ZINA (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
The small mitogenic cytokine Pleiotrophin (PTN) is well-known for its roles in
tissue growth, development, and repair. First isolated from neuronal tissues, much interest in this protein resides in development of the central nervous system and neuronal regeneration. Owning to its role in growth, development and its ability to promote angiogenesis and metastasis, PTN’s overexpression in cancers such as glioblastoma, has become the focal point of much research. Many of the receptors through which PTN acts contain glycosaminoglycans (GAGs), through which PTN binds. Thus, understanding the atomistic detail of PTN’s architecture and interaction with GAG chains is of significant importance in elucidating its functional role in growth and malignancy of biological tissues, as well as in neural development and progression of other diseases. Herein the first solution state structure of PTN was solved via nuclear magnetic resonance (NMR), with extensive characterization of its ability to bind GAG. Structurally, PTN consists of two -sheet domains connected by a short flexible linker, and flanked by long flexible termini. Broad distribution of positively charged amino acids in the protein’s sequence yields highly basic surfaces on the -sheet domains as well as highly cationic termini. With GAG chains themselves being linear anionic polymers, all interactions between these sugars and PTN are most exclusively driven through the electrostatic interactions between them, with no discernable specificity for GAG types. Moreover, this binding event is coordinated mostly through basic patches located in the C-Terminal domain (CTD). Although the flexible C- terminus has been shown to play a significant role in receptor binding, data here also reveal an adaptability of PTN to maintain high affinity interactions through its structured domains
when termini are removed. Additionally, analysis of binding information revealed for the first time the presence of a secondary GAG binding site within PTN. It is shown that PTN’s CTD constitutes the major binding site, while the N-terminal domain (NTD) contains the much weaker secondary site. Finally, compilation of high-resolution data containing the atomistic detail of PTN’s interaction with GAG provided the information necessary to produce the highest accuracy model to date of the PTN-GAG complex. Taken together, these findings provide means for specific targeting of this mitogenic cytokine in a wide array of biological applications.
tissue growth, development, and repair. First isolated from neuronal tissues, much interest in this protein resides in development of the central nervous system and neuronal regeneration. Owning to its role in growth, development and its ability to promote angiogenesis and metastasis, PTN’s overexpression in cancers such as glioblastoma, has become the focal point of much research. Many of the receptors through which PTN acts contain glycosaminoglycans (GAGs), through which PTN binds. Thus, understanding the atomistic detail of PTN’s architecture and interaction with GAG chains is of significant importance in elucidating its functional role in growth and malignancy of biological tissues, as well as in neural development and progression of other diseases. Herein the first solution state structure of PTN was solved via nuclear magnetic resonance (NMR), with extensive characterization of its ability to bind GAG. Structurally, PTN consists of two -sheet domains connected by a short flexible linker, and flanked by long flexible termini. Broad distribution of positively charged amino acids in the protein’s sequence yields highly basic surfaces on the -sheet domains as well as highly cationic termini. With GAG chains themselves being linear anionic polymers, all interactions between these sugars and PTN are most exclusively driven through the electrostatic interactions between them, with no discernable specificity for GAG types. Moreover, this binding event is coordinated mostly through basic patches located in the C-Terminal domain (CTD). Although the flexible C- terminus has been shown to play a significant role in receptor binding, data here also reveal an adaptability of PTN to maintain high affinity interactions through its structured domains
when termini are removed. Additionally, analysis of binding information revealed for the first time the presence of a secondary GAG binding site within PTN. It is shown that PTN’s CTD constitutes the major binding site, while the N-terminal domain (NTD) contains the much weaker secondary site. Finally, compilation of high-resolution data containing the atomistic detail of PTN’s interaction with GAG provided the information necessary to produce the highest accuracy model to date of the PTN-GAG complex. Taken together, these findings provide means for specific targeting of this mitogenic cytokine in a wide array of biological applications.
ContributorsRyan, Eathen (Author) / Wang, Xu (Thesis advisor) / Yarger, Jeffery (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020