ENZYME-FREE UBIQUITIN LIGATION. FROM NATIVE CHEMICAL LIGATION AND SPIN LABELING TO DIMERIZATION BY INTER-UBIQUITIN MIMICKING LINKAGE AND PH-DEPENDENT CONFORMATIONAL SWITCH

The delicate balance between the production and disposal of proteins is vital for the changes required in the cell to respond to given stimulus. Ubiquitination is a protein modification with a range of signaling outcomes when ubiquitin is attached to a protein through a highly ordered enzymatic cascade process. Understanding ubiquitination is a growing field and nowadays the application of chemical reactions allows the isolation of quantitative materials for structural studies. Therefore, in this dissertation it is described some of these suitable chemical methodologies to produce an isopeptide bond toward the polymerization of ubiquitin bypassing the enzymatic control with the purpose of showing if these chemical modifications have a direct impact on the structure of ubiquitin. First, the possibility of incorporating non-natural lysine analogs known as mercaptolysines into the polypeptide chain of Ubiquitin was explored when they were attached to ubiquitin by native chemical ligation at its C terminus. The sulfhydryl group was used for the attachment of a paramagnetic label to map the surface of ubiquitin. Second, the condensation catalyzed by silver nitrate was used for the dimer assembly. In particular, the main focus was on examining whether orthogonal protection and deprotection of each monomer have an impact on the reaction yield, since the synthetic strategy has been previously attempted successfully. Third, the formation of ubiquitin dimers was approached by building an inter-ubiquitin linkage mimicking the isopeptide bond with two approaches, the classic disulfide exchange as well as the thiol-ene click reaction by thermal initiation in aqueous conditions. After assembling the dimeric units, they were studied by Nuclear Magnetic Resonance, in order to establish a conformational state profile which depends on the pH conditions. The latter is a very important concept since some ligands have a preferred affinity when the protein-protein hydrophobic patches are in close proximity.

SOCIAL STRUCTURE OF ASIAN ELEPHANTS (ELEPHAS MAXIMUS) IN SRI LANKA

Asian elephants (Elephas maximus) are critically endangered and live in fragmented populations spread across 13 countries. Yet in comparison to the African savannah elephant (Loxodonta africana), relatively little is known about the social structure of wild Asian elephants because the species is mostly found in low visibility habitat. A better understanding of Asian elephant social structure is critical to mitigate human-elephant conflicts that arise due to increasing human encroachments into elephant habitats. In this dissertation, I examined the social structure of Asian elephants at three sites: Yala, Udawalawe, and Minneriya National Parks in Sri Lanka, where the presence of large open areas and high elephant densities are conducive to behavioral observations. First, I found that the size of groups observed at georeferenced locations was affected by forage availability and distance to water, and the effects of these environmental factors on group size depended on site. Second, I discovered that while populations at different sites differed in the prevalence of weak associations among individuals, a core social structure of individuals sharing strong bonds and organized into highly independent clusters was present across sites. Finally, I showed that the core social structure preserved across sites was typically composed of adult females associating with each other and with other age-sex classes. In addition, I showed that females are social at all life stages, whereas males gradually transition from living in a group to a more solitary lifestyle. Taking into consideration these elements of Asian elephant social structure will help conservation biologists develop effective management strategies that account for both human needs and the socio-ecology of the elephants.

STRUCTURE AND FUNCTION OF THE GROUP III CHAPERONINS, A UNIQUE CLADE OF PROTEIN FOLDING NANOMACHINES

The survival and descent of cells is universally dependent on maintaining their proteins in a properly folded condition. It is widely accepted that the information for the folding of the nascent polypeptide chain into a native protein is encrypted in the amino acid sequence, and the Nobel Laureate Christian Anfinsen was the first to demonstrate that a protein could spontaneously refold after complete unfolding. However, it became clear that the observed folding rates for many proteins were much slower than rates estimated in vivo. This led to the recognition of required protein-protein interactions that promote proper folding. A unique group of proteins, the molecular chaperones, are responsible for maintaining protein homeostasis during normal growth as well as stress conditions. Chaperonins (CPNs) are ubiquitous and essential chaperones. They form ATP-dependent, hollow complexes that encapsulate polypeptides in two back-to-back stacked multisubunit rings, facilitating protein folding through highly cooperative allosteric articulation. CPNs are usually classified into Group I and Group II. Here, I report the characterization of a novel CPN belonging to a third Group, recently discovered in bacteria. Group III CPNs have close phylogenetic association to the Group II CPNs found in Archaea and Eukarya, and may be a relic of the Last Common Ancestor of the CPN family. The gene encoding the Group III CPN from Carboxydothermus hydrogenoformans and Candidatus Desulforudis audaxviator was cloned in E. coli and overexpressed in order to both characterize the protein and to demonstrate its ability to function as an ATPase chaperone. The opening and closing cycle of the Chy chaperonin was examined via site-directed mutations affecting the ATP binding site at R155. To relate the mutational analysis to the structure of the CPN, the crystal structure of both the AMP-PNP (an ATP analogue) and ADP bound forms were obtained in collaboration with Sun-Shin Cha in Seoul, South Korea. The ADP and ATP binding site substitutions resulted in frozen forms of the structures in open and closed conformations. From this, mutants were designed to validate hypotheses regarding key ATP interacting sites as well as important stabilizing interactions, and to observe the physical properties of the resulting complexes by calorimetry.

Changes in the community structure of urban and rural forest patches in Baltimore from 1998 to 2015

Urban forests are often highly fragmented with many exotic species. Altered disturbance regimes and environmental pollutants influence urban forest vegetation. One of the best ways to understand the impacts of land-use on forest composition is through long-term research. In 1998, the Baltimore Ecosystem Study established eight forest plots to investigate the impacts of urbanization on natural ecosystems. Four plots were located in urban forest patches and four were located in rural forests. In 2015, I revisited these plots to measure abundances and quantify change in forest composition, diversity, and structure. Sapling, shrub, and seedling abundance were reduced in the rural plots. Alpha diversity and turnover was lower in the rural plots. Beta diversity was reduced in the rural plots. The structure of the urban plots was mostly unchanged, except for a highly reduced sapling layer. Beta diversity in the urban plots was consistent across surveys due to high species turnover.