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Description
Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with macromolecular crystallography, primarily the difficulty of growing large, well-ordered protein crystals. Since the first proof of concept for femtosecond nanocrystallography showing that diffraction patterns can be collected on extremely small crystals, thus negating the need to grow larger crystals, there have been many exciting advancements in the field. The technique has been proven to show high spatial resolution, thus making it a viable method for structural biology. However, due to the ultrafast nature of the technique, which allows for a lack of radiation damage in imaging, even more interesting experiments are possible, and the first temporal and spatial images of an undamaged structure could be acquired. This concept was denoted as time-resolved femtosecond nanocrystallography.
This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
ContributorsKupitz, Christopher (Author) / Fromme, Petra (Thesis advisor) / Spence, John C. (Thesis advisor) / Redding, Kevin (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2014
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
X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm – ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm – ~20 μm crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion. Additionally, a passive mixer was created to generate unique solution concentrations within isolated nanowells to crystallize phycocyanin and lysozyme. Crystal imaging with brightfield microscopy, UV fluorescence, and SONICC coupled with numerical modeling allowed quantification of crystal growth conditions for efficient phase diagram development. The developed microfluidic tools demonstrated the capability of improving samples for protein crystallography, offering a foundation for continued development of platforms to aid protein structure determination.
ContributorsAbdallah, Bahige G (Author) / Ros, Alexandra (Thesis advisor) / Buttry, Daniel (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2016
Description
Protein crystallization has become an extremely important tool in biochemistry since the first structure of the protein Myoglobin was solved in 1958. Survival of motor neuron protein has proved to be an elusive target in regards to producing crystals of sufficient quality for X-ray diffraction. One form of Survival of motor neuron protein has been found to be a cause of the disease Spinal Muscular Atrophy that currently affects 1 in 6000 live births. The production, purification and crystallization of Survival of motor neuron protein are detailed. The Fenna-Matthews-Olson (FMO) protein from Pelodictyon phaeum is responsible for the transfer of energy from the chlorosome complex to the reaction center of the bacteria. The three-dimensional structure of the protein has been solved to a resolution of 2.0Å with the Rwork and Rfree values being 16.6% and 19.9% respectively. This new structure is compared to the FMO protein structures of Prosthecocholoris aestuarii 2K and Chlorobium tepidum. The early structures of FMO contained seven bacteriochlorophyll-a (BChl) molecules but the recent discovery that there is an eighth BChl molecule in Ptc. aestuarii 2K and Cbl. tepidum and now in Pld. phaeum requires that the energy transfer mechanism be reexamined. Simulated spectra are fitted to the experimental optical spectra to determine how the BChl molecules transfer energy through the protein. The inclusion of the eighth BChl molecule within these simulations may have an impact on how energy transfer through FMO can be described. In conclusion, a reliable method of purifying and crystallizing the SMNWT protein is detailed, the placement of the 8th BChl-a within the electron density and the implications on energy transfer within the FMO protein when the 8th BChl-a is included from the green sulfur bacteria Pld. phaeum is discussed.
ContributorsLarson, Chadwick R (Author) / Allen, James P. (Thesis advisor) / Francisco, Wilson (Committee member) / Chen, Julian (Committee member) / Arizona State University (Publisher)
Created2010
Description
DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.
ContributorsHostal, Anna Elizabeth (Author) / Anderson, Karen (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Yan, Hao (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
With the rise of global warming and the growing energy crisis, scientists have pivoted from typical resources to look for new materials and technologies that can aid in advancing renewable energy efforts. Perovskite materials hold the potential for making high-efficiency, low-cost solar cells through solution processing of Earth abundant materials; however, scalability and manufacturability remain a challenge. In order to transition from small scale processing in inert environments via spin coating to higher throughput processing in ambient conditions via blade coating, the fundamentals of perovskite crystallization must be understood. Classical nucleation theory, the LaMer relation, and nonclassical crystallization considerations are discussed to provide a mechanism by which gellan gum, a nontoxic biopolymer from the food industry, has enabled quality halide perovskite thin films. Specifically, this research aims to study the effects of gellan gum in improving perovskite manufacturability by controlling crystallization through indirect alteration of evaporation and supersaturation rates by modifying fluid dynamics and the free energy associated with nucleation and growth. Simply, gellan gum controls crystallization to enable the fabrication of promising scalable PVSK devices in open air.
ContributorsCartledge, Carsen (Author) / Rolston, Nicholas (Thesis director) / Yu, Zhengshan (Committee member) / Barrett, The Honors College (Contributor) / Materials Science and Engineering Program (Contributor) / School of International Letters and Cultures (Contributor) / School of Art (Contributor)
Created2023-05
Description
Since its implementation in 1966, the National Historic Preservation Act (NHPA) has greatly informed preservation practice in the United States. As a primary text for the professionalization of the field of preservation, it not only acts as a law, but establishes an ideological framework that informs practices which impact public memory in the US by determining what places remain, how they are transformed (or not), and whose stories they tell. The objective of this study is to explore the communicative dimensions of the NHPA to better understand how its rhetoric informs practice, and thus, informs public memory in the US. This study employs a meta-method of crystallization which engages a range of analysis methods. First, I conducted a close rhetorical analysis of the NHPA’s text which provided insight into ideologies within the law, opportunities for practice, and limitations on practice through the law’s definitive conceptualization of public memory. Next, I completed a qualitative case study of a preservation organization. I participated in extended field observation, conducted interviews with organizational staff, and engaged in walking methods in the city. The analysis offered insight into local discourses (everyday talk) which built into Discourses (ideologies) and demonstrated how the NHPA informs d/Discourses of preservation, even when it is not required. Although local practice was informed by the NHPA, the analysis also revealed methods for challenging and resisting the NHPA. Finally, I engaged in arts-based methods to examine how National Register listings (products of the NHPA) provide aesthetic and narrative precedents for determining ‘significance’ and worth in preservation practice. Through a poetic exhibition entitled Mythed Places, I artistically analyzed the NRHP, arguing that by giving historic sites the quality of myths, the NHPA attempts to arrest multiple unfolding narratives of places in service of a national myth.
This project demonstrates that the NHPA communicatively constructs US preservation practice through its ambiguity, implied morality, and formation of a mythic national community. Although the current structure requires preservationists, even those not legally bound to the NHPA, to work within its framework, this study showcases ways to disrupt the discursive boundaries and practice preservation more critically.
ContributorsWheeler, Ashley (Author) / Brouwer, Daniel C (Thesis advisor) / Zanin, Alaina C (Committee member) / McHugh, Kevin E (Committee member) / Arizona State University (Publisher)
Created2021