ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
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- All Subjects: Biochemistry
- Creators: Liu, Wei
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.
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.
The studies conducted in this dissertation probed the role of the S1-S4 membrane domain in temperature and ligand activation of human TRPV1. Temperature-dependent solution nuclear magnetic resonance (NMR) spectroscopy for thermodynamic and mechanistic studies of the S1-S4 domain. From these results, a potential temperature sensing mechanism of TRPV1, initiated from the S1-S4 domain, was proposed. Additionally, direct binding of various ligands to the S1-S4 domain were used to ascertain the interaction site and the affinities (Kd) of various ligands to this domain. These results are the first to study the isolated S1-S4 domain of human TRPV1 and many results indicate that the S1-S4 domain is crucial for both temperature-sensing and is the general receptor binding site central to chemical activation.
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.
Inspired by the successful serial crystallography (SX) experiment at a synchrotron radiation source, it is first-time equipping the high-viscosity injector to X-ray fluxes increased at 100 times by a moderate increased in bandwidth to perform the pink beam SX experiments. The structure of proteinase K (PK) was determined to 1.8 Å resolution with 4 consecutive 100 ps X-ray pink beam pulse exposures. The structure of human A2A adenosine receptor (A2AAR) reached to a 4.2 Å resolution using 24 consecutive X-ray pink beam pulse exposures. It has proven the feasibility to utilize such storage-ring synchrotron sources complemented to serial femtosecond crystallography, presenting new opportunities for microcrystallography and the time-resolved experiments.
As an alternative approach to serial femtosecond crystallography, a novel protocol was developed to combine the lipidic cubic phase crystallization approach and microED strategy and solved the structure from LCP-embedded proteinase K microcrystals with the comparable high resolution to conventional crystallographic method.
It cannot be neglected that only very few portions of membrane proteins were able to be successfully crystallized for structure determination. Single particle cryoEM method allows the structural studies from protein molecules detour away from crystallization. An atomic resolution structure of the β1-AR bound with agonist in complex with Gs protein, with particle size of less than 200 kDa, was determined by cryoEM, reaching to an atomic resolution of 3.8 Å. The complex structure captured a fully active conformation and revealed the important mechanisms of how the agonist bound receptor activated Gs protein.
These technological developments provide more opportunities to the structural biology community to discover mechanisms underlying such complicated machinery network, which would eventually benefit the structure-based drug discovery.
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.