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: Mills, Jeremy
This thesis focused on the expression and crystallization the fragment antigen binding antibody fragment A4. A fragment antigen binding fragment was chosen to be worked with as it is more stable than many other antibody fragments. A4 is important in Alzheimer’s disease as it is able to identify toxic beta amyloid.
I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.
SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.
Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.
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.