Matching Items (4)
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- All Subjects: Crystallography
- Creators: Levitus, Marcia
- Creators: Benn-Torres, Jada
- Member of: ASU Electronic Theses and Dissertations
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
ContributorsHunter, Mark (Author) / Fromme, Petra (Thesis advisor) / Wolf, George (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
Although the Caribbean has been continuously inhabited for the last 7,000 years, European contact in the last 500 years dramatically reshaped the cultural and genetic makeup of island populations. Several recent studies have explored the genetic diversity of Caribbean Latinos and have characterized Native American variation present within their genomes. However, the difficulty of obtaining ancient DNA from pre-contact populations and the underrepresentation of non-Latino Caribbean islanders in current research have prevented a complete understanding of genetic variation over time and space in the Caribbean basin. This dissertation uses two approaches to characterize the role of migration and admixture in the demographic history of Caribbean islanders. First, autosomal variants were genotyped in a sample of 55 Afro-Caribbeans from five islands in the Lesser Antilles: Grenada, St. Kitts, St. Lucia, Trinidad, and St. Vincent. These data were used to characterize genetic structure, ancestry and signatures of selection in these populations. The results demonstrate a complex pattern of admixture since European contact, including a strong signature of sex-biased mating and inputs from at least five continental populations to the autosomal ancestry of Afro-Caribbean peoples. Second, ancient mitochondrial and nuclear DNA were obtained from 60 skeletal remains, dated between A.D. 500–1300, from three archaeological sites in Puerto Rico: Paso del Indio, Punta Candelero and Tibes. The ancient data were used to reassesses existing models for the peopling of Puerto Rico and the Caribbean and to examine the extent of genetic continuity between ancient and modern populations. Project findings support a largely South American origin for Ceramic Age Caribbean populations and identify some genetic continuity between pre and post contact islanders. The above study was aided by development and testing of extraction methods optimized for recovery of ancient DNA from tropical contexts. Overall, project findings characterize how ancient indigenous groups, European colonial regimes, the African Slave Trade and modern labor movements have shaped the genomic diversity of Caribbean islanders. In addition to its anthropological and historical importance, such knowledge is also essential for informing the identification of medically relevant genetic variation in these populations.
ContributorsNieves Colón, Maria (Author) / Stone, Anne C (Thesis advisor) / Pestle, William J. (Committee member) / Benn-Torres, Jada (Committee member) / Stojanowski, Christopher (Committee member) / Arizona State University (Publisher)
Created2017
Description
Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points (𝑡"), and current sample delivery methods have complex fabrication and assembly requirements.
To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.
For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.
To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.
For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.
ContributorsEchelmeier, Austin (Author) / Ros, Alexandra (Thesis advisor) / Levitus, Marcia (Committee member) / Weierstall, Uwe (Committee member) / Arizona State University (Publisher)
Created2019