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Gle1 is an mRNP export mediator with major activity localized to the nuclear pore complex in eukaryotic cells. The protein's high preservation across vast phylogenetic distances allows us to approximate research on the properties of yeast Gle1 (yGle1) with those of human Gle1 (hGle1). Research at Vanderbilt University in 2016, which provides the research basis of this thesis, suggests that the coiled-coil domain of yGle1 is best crystallized in dicationic aqueous conditions of pH ~8.0 and 10\u201420% PEG 8000. Further exploration of crystallizable microconditions revealed a favorability toward lower pH and lower PEG concentration. Following the discovery of the protein's native crystallography conditions, a comprehensive meta-analysis of scientific literature on Gle1 was conducted on the association of Gle1 mutations with neuron disease.
ContributorsGaetano, Philip Pasquale (Author) / Foy, Joseph (Thesis director) / Dawson, T. Renee (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The dopamine 2 receptor (D2R) is a Class A GPCR which is essential for signaling in the nervous system, and has been implicated in numerous illnesses. While there are over 50 currently approved drugs which act on D2R, the structure has never been determined in detail. Although crystallography has historically been difficult with GPCRs, in recent years many structures have been solved using lipidic cubic phase (LCP) crystallization techniques. Sample preparation for LCP crystallization typically requires optimization of genetic constructs, recombinant expression, and purification techniques in order to produce a sample with sufficient stability and homogeneity. This study compares several genetic constructs utilizing different promoters, fusion proteins, fusion positions, and truncations in order to determine a high quality construct for LCP crystallization of
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.
ContributorsErler, Maya Marie (Author) / Liu, Wei (Thesis director) / He, Ximin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Developments in structural biology has led to advancements in drug design and vaccine development. By better understanding the macromolecular structure, rational choices can be made to improve factors in such as binding affinity, while reducing promiscuity and off-target interactions, improving the medicines of tomorrow. The majority of diseases have a macromolecular basis where rational drug development can make a large impact. Two challenging protein targets of different medical relevance have been investigated at different stages of determining their structures with the ultimate goal of advancing in drug development. The first protein target is the CapBCA membrane protein complex, a virulence factor from the bacterium Francisella tularensis and the causative agent of tularemia and classified as a potential bioterrorism weapon by the United States. Purification of the individual protein targets from the CapBCA complex is a key and challenging step that has been, so far, a limiting factor towards the structure determination of the whole complex. Here, the purification protocols for the CapB and CapC subunits have been establish, which will allow us to progress towards biophysical and structural studies. The second protein target investigated in this thesis is the catalytically active Taspase1. Taspase1 functions as a non-oncogene addiction protease that coordinates cancer cell proliferation and apoptosis and has been found to be overexpressed in many primary human cancers. Here the structure is presented to 3.04A with the goal of rational drug design of Taspase1 inhibitors. Development of Taspase1 inhibitors has no completion in the drug discovery arena and would function as a new anti-cancer therapeutic. Solving the structures of medically relevant proteins such as these is critical towards rapidly developing treatments and prevention of old and new diseases.
ContributorsJernigan, Rebecca J. (Author) / Fromme, Petra (Thesis director) / Hansen, Debra T. (Committee member) / Martin-Garcia, Jose M. (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Protein crystallization is a technique for the formation of three-dimensional protein crystals, which is widely utilized by scientists, engineers, and researchers. Protein crystallography allows for protein structures and functions to be studied. As proteins play a central role in biological systems and life itself, a deeper understanding of their structure-function properties is crucial to elucidating fundamental behaviors, such as protein folding in addition to the role that they play in emerging fields, such as, tissue engineering with application to the emerging field of regenerative medicine. However, a significant limitation toward achieving further advancements in this field is that in order to determine detailed structure of proteins from protein crystals, high-quality and larger size protein crystals are needed. Because it is difficult to produce adequate size, high-quality crystals, it remains difficult to determine the structure of many proteins. However, a new method using a microgravity environment to crystallize proteins has proven effective through various studies conducted on the International Space Station (ISS). In the presence of microgravity, free convection is essentially absent in the bulk solution where crystallization occurs, thus allowing for purely random Brownian motion to exist which favors the nucleation and growth of high-quality protein crystals. Many studies from the ISS to date have demonstrated that growing protein crystals in a microgravity environment produces larger and higher-quality crystals. This method provides new opportunities for better structure identification and analysis of proteins. Although there remains many more limitations and challenges in the field, microgravity protein crystallization holds many opportunities for the future of biotechnology and scientific development. The objective of this thesis was to study the crystallization of hen egg white lysozyme (HEWL) and determine the effects of both unit and microgravity on growth/size and quality of HEWL. Through preliminary trials using a universal ground-based reduced-gravity system, the crystallization of HEWL in a simulated microgravity environment was successfully conducted and the results reported are promising. The utility of continuous, scalable ground-based, microgravity platforms for studies on a wide range of material systems and behavior, such as, protein crystallization, has significant implications regarding its impact on many industries, including drug development as well as regenerative medicine.
ContributorsTran, Amanda Marie (Author) / Pizziconi, Vincent (Thesis director) / Alford, Terry (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-12