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.

DNA nanotechnology uses the reliability of Watson-Crick base pairing to program and generate two-dimensional and three-dimensional nanostructures using single-stranded DNA as the structural material. DNA nanostructures show great promise for the future of bioengineering, as there are a myriad of potential applications that utilize DNA’s chemical interactivity and ability to bind other macromolecules and metals. DNA origami is a method of constructing nanostructures, which consists of a long “scaffold” strand folded into a shape by shorter “staple” oligonucleotides. Due to the negative charge of DNA molecules, divalent cations, most commonly magnesium, are required for origami to form and maintain structural integrity. The experiments in this paper address the discrepancy between salt concentrations required for origami stability and the salt concentrations present in living systems. The stability of three structures, a two-dimensional triangle, a three-dimensional solid cuboid and a three-dimensional wireframe icosahedron were examined in buffer solutions containing various concentrations of salts. In these experiments, DNA origami structures remained intact in low-magnesium conditions that emulate living cells, supporting their potential for widespread biological application in the future.

Microsolvation studies have begun to shed the light on the impact that single water molecules have on the structure of a molecule. The difference in behavior that molecules show when exposed to an increasing number of water molecules has been considered important but remains elusive. The cluster distributions of formic acid were studied for its known importance as an intermediate in the water gas shift reaction. Implementations of the water gas shift reaction range from a wide range of applications. Studies have proposed implementations such as variety such as making water on the manned mission to mars and as an industrial energy source. The reaction pathway of formic acid favors decarboxylation in solvated conditions but control over the pathway is an important field of study. Formic acid was introduced into a high vacuum system in the form of a cluster beam via supersonic expansion and was ionized with the second harmonic (400nm) of a pump-probe laser. Mass spectra showed a ‘magic’ 5,1 (formic acid, water) peak which showed higher intensity than was usually observed in clusters with 1 water molecule. Peak integration showed a higher relative abundance for the 5,1 cluster as well and showed the increased binding favorability of this conformation. As a result, there is an enhanced probability of molecules sticking together in this arrangement and this is due to the stable, cage-like structure that the formic acid forms when surrounding the water molecule.

Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than deoxyribose nucleic acid (DNA) sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. In 2011, Haynes et al. showed that a synthetic regulator called the Polycomb chromatin Transcription Factor (PcTF), a fusion protein that binds methylated histones, reactivated an artificially-silenced luciferase reporter gene. These synthetic transcription activators are derived from the polycomb repressive complex (PRC) and associate with the epigenetic silencing mark H3K27me3 to reactivate the expression of silenced genes. It is demonstrated here that the duration of epigenetic silencing does not perturb reactivation via PcTF fusion proteins. After 96 hours PcTF shows the strongest reactivation activity. A variant called Pc2TF, which has roughly double the affinity for H3K27me3 in vitro, reactivated the silenced luciferase gene by at least 2-fold in living cells.

In the past decade, technological breakthroughs have facilitated structure determination of so many difficult-to-study membrane protein targets. In this thesis research, three techniques were investigated to enable the structural determination of such challenging targets, polychromatic pink-beam serial crystallography with high-viscous sample, lipidic cubic phase (LCP)-based microcrystal electron diffraction (MicroED), and single-particle cryogenic electron microscopy targeting (cryoEM).

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.

Mass nuclear catastrophe is a serious concern for society at large when considering the rising threat of terrorism and the risks associated with harnessing nuclear energy. In the case of a mass nuclear/radiological event that requires hundreds of thousands of individuals to be assessed for radiation exposure, a rapid biodosimetry triage tool is crucial [1]. The Cytokinesis Block Micronucleus Assay (CBMN) is a promising cytogenetic biodosimetry assay for triage [2]; however, it requires shipping samples to a central laboratory (1-3 days) followed by a lengthy cell culture process (~3 days) before the first dose estimate can be available. The total ~ 1 week response time is too long for effective medical care intervention. A shipping incubator could cut the response time in half (~3 days) by culturing samples in transit; however, possible shipping delays beyond 2 days without the addition of a necessary reagent (Cyto-B) would ruin the integrity of the samples—for accurate CBMN assay endpoint observation, Cyto-B must be added within a 24-44 hour window after sample culture is initiated. Here, we propose a “Smart” Shipping Incubator (SSI) that can add Cyto-B while samples are in transit through a centrifugal system equipped with microfluidic capillary valve caps. The custom centrifugal system was constructed with CNC machined and 3D printed plastic parts, controlled by a custom printed circuit board (PBC) microcontroller, and housed inside a commercial shipping incubator (iQ5 from MicroQ Technologies). Teflon-coated, pre-pulled glass micropipettes (FivePhoton BioChemicals) were used as microfluidic capillary valve caps. Release of Cyto-B was characterized by a desktop centrifugal system at different tip sizes and relative centrifugal forces (RCFs). A theoretical model of Cyto-B release was also deduced to aid the optimization of the process. The CBMN assay was conducted both in the SSI with centrifugal Cyto-B release and in a standard CO2 incubator with manual addition of Cyto-B as the control. The expected mechanical shock during shipment was measured to be less than 25g. Optimal Cyto-B release was found to be at 35g RCF with a Teflon-coated 40 µm tip. Similar CBMN dose curves of micronuclei per binucleated cells (MN/BN) vs. exposed radiation (Gy) were produced for samples assessed conventionally and with the SSI. The similarities between the two methods suggest that centrifugation does not significantly affect the CBMN assay.

Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin.

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.

Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors.

Abstract: It has been established that α-keto-analogs of amino acids can be converted into the amino acids through transamination in vivo. This discovery led to breakthroughs in treating patients who had difficulty digesting traditional proteins, such as in chronic kidney disease (CKD) sufferers where patients have poor kidney function, which poisons the blood with ammonia products.
This pilot study aimed to ascertain the potential for keto acid supplementation in the attempt to supply adequate protein building blocks to healthy populations, with the caveats that said supplementation 1) would utilize non-synthetic methods, 2) offer an alternative to high-phosphate protein supplies such as ruminant animals, and 3) reverse the ill effects of ammonia load by reducing nitrogen intake and consuming ammonia as a fuel for the process of protein synthesis. This proposed solution turns to orange juice and certain varietals of potato juice for their familiarity to consumers, innate nutritional values, and potential for mass-production by many existing companies. The work contained here represents the first phase of experimentation: qualifying the presence of α-keto-analogues of amino acids in these types of produce which, with transamination, could yield the amino acids necessary for adequate protein intake.
Results suggest that these juices do not contain adequate α-keto-analogs of amino acids to supplement proteins in either healthy or ill individuals.

2,2’ bipyridine (Bpy) can form metal complexes with divalent metals in the form of [M(Bpy-ala)¬3]+2 where M is any divalent metal. These [M(Bpy-ala)¬3]+2 complexes can have very interesting photochemical and redox potentials that can be useful in more complex systems. The use of (2,2′-bipyridin-5yl)alanine (Bpy-ala) as a Noncanonical Amino Acid (NCAA) has allowed Bpy to be incorporated into an amino acid sequence which can now function in a protein scaffold. Previous studies have utilized that power of Bpy-ala to design a protein that can assemble a homotrimeric protein complex in the presence of a divalent metal. However, the issue with this design was that when the homotrimer was formed and the divalent was removed, the protein complex would not dissemble indicating that it was not metal dependent. Point mutations were made to disrupt the protein-protein interactions to favor disassembly in the absence of a divalent metal. Successfully, a mutation was made that allowed the designed protein to be metal dependent for self-assembly. Nevertheless, an issue with this design is that it poorly incorporated ruthenium(II) into the tris Bpy complex forming [Ru(Bpy-ala)¬3]+2, which was one of the main goals of the original design. This thesis sets out to form TRI 05 I13S M6I which should uphold the same metal-dependence as its predecessor and should combine ruthenium (II) into the protein complex forming [Ru(Bpy-ala)¬3]+2. The thesis shows the success of formation and expression of TRI 05 I13S M6I in Escherichia coli cells. This thesis also reports several purification steps and procedures to not only purify TRI 05 I13S M6I but also removing both the His-tag sequence and Fe(II) from the protein. The thesis also shows that TRI 05 I13S M6I does not behave like its predecessor in that it is not metal dependent for self-assembly. While this may be true, this paper also reports the incorporation of ruthenium (II) in the protein structure. Though this may be the first time that ruthenium (II) has been recorded to be in the TRI 05 protein complex with a significant signal, it is still nowhere near the optimal fluorescence that small molecule Bpy can achieve by itself. The thesis reports potential conditions and a plan of attack that should drive this project forward into achieving an optimal signal of the [Ru(Bpy-ala)¬3]+2 complex in a TRI 05 protein scaffold.