Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 161 p.; also includes graphics (some col.) Includes bibliographical references (p. 144-161). Available online via OhioLINK's ETD Center

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed April 27, 2010). Thesis advisor: Dr. Kent S. Gates. Vita. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / Fast photochemical oxidation of proteins (FPOP) is an emerging footprinting method that utilizes hydroxyl radicals. The use of hydroxyl radicals create stable labeled products that can be analyzed with mass spectrometry. The advantage of FPOP over other methods is the fast acquisition of results and the small amount of sample required for analysis. Protein structure and protein- ligand interactions have been studied with FPOP. Here we evaluated (1) the reproducibility of FPOP, (2) the effect of hydrogen peroxide concentration on oxidation and (3) the use of FPOP to evaluate protein- nucleic acid interaction with Apurinic/Apurinic endonuclease 1 (APE1) protein. APE1 is a pleotropic protein that has been crystallized and studied widely. The 35641.5 Da protein has two major functional activities: DNA repair and redox function. An intact protein study of APE1 showed consistent global labeling by FPOP and a correlation between oxidation and hydrogen peroxide concentration. Furthermore, analysis of APE1 with DNA was done in hopes of probing the DNA binding site. Although the oxidation observed was not sufficient to define the complex pocket, a dramatic effect was seen in residue oxidation when DNA was added. Interestingly, the internal residues were labeled collectively in all APE1 experiments which indicates partial unfolding of the protein as previously suggested in the literature. Hence, these findings establish the use of FPOP to capture protein dynamics and provide evidence of the existence breathing dynamics of APE1.

This thesis describes work done to design and synthesize protease inhibitors, with the intention of developing therapeutic agents for Alzheimer’s disease (AD) and the chronic liver condition caused by infection of the hepatitis C virus (HCV). AD is the most common form of dementia, and HCV infection is the primary reason for liver transplantation in industrialized countries. Today, these two illnesses affect 24 and 170 million people, respectively. It has been shown that the human aspartic protease BACE-1 plays an important role in the development of AD, and thus inhibition of BACE-1 may offer a way to improve the quality of life of individuals afflicted with the disease. Furthermore, it is known that the serine protease NS3 is a vital component in the replication of HCV. Several novel potent BACE-1 inhibitors encompassing different transition state mimics were prepared. First, a hydroxyethylene moiety encompassing a secondary hydroxyl group was evaluated as a transition state analogue, producing inhibitors in the low nanomolar range. Various tertiary hydroxyl isosteres were also investigated as the central core, with the aim of shielding the pivotal hydroxyl group. These transition state isosteres consisted of tertiary hydroxyl analogues of previously used secondary hydroxyl containing norstatine, statine, and hydroxyethylamine isosteres. Several tertiary hydroxyl-containing inhibitors were found to be active in the low micromolar range. In addition, two inhibitors were co-crystallized with the BACE-1 enzyme to provide X-ray crystal structures, which furnished valuable binding information for further design of improved BACE-1 inhibitors. The goal in the HCV NS3 protease inhibitor project was to design, synthesize and evaluate a novel hydroxycyclopentene bioisostere to the previously used acyl-hydroxyproline moiety. The investigation revealed that it was possible to synthesize inhibitors containing this new bioisostere that were potent in the low nanomolar range. Further optimization by rigidification of the most active inhibitor resulted in equipotent macrocyclic compounds.

This thesis studies hydrated oxygen and hydroxyl radicals as a basis for understanding the species formed in the icy surfaces of outer solar system bodies. Infrared spectroscopy is used to identify the species water-oxygen (H2O·O2) and water-hydroxyl (H2O·HO) complexes in inert gas matrices and presents a new mechanism for O2 formation in irradiated ices. The H2O·O2 Complex -- The H2O·O2 complex was identified in solid argon matrices at 11 K by measuring the infrared spectra of H2O⁄O2⁄Ar matrices. Absorption bands at 3731.6, 3638.3, 1590.2⁄1593.6 and 1551.9⁄1548.8 cm-1 were respectively assigned to asymmetric OH water stretching, symmetric OH water stretching, H2O bending, and the O2 stretching vibrations. This experimental data was in good agreement with the results of quantum mechanical calculations that predict the vibrational frequencies and intensities for H2O·O2. These calculations gave a binding energy of 0.72 kcal mol-1 for the complex. The H2O·HO Complex -- The H2O·HO complex was identified in solid argon matrices at 11 K by measuring the infrared spectra of OH⁄H2O⁄Ar matrices. The OH was formed in a Tesla coil discharge of an H2O⁄Ar gas stream. This gas stream also provided the source of H2O and Ar needed for the experiments. Three absorption bands were assigned to the OH stretch of the hydroxyl group in the complex. These three bands were caused by the occupancy of three different lattice sites. This experimental data was in good agreement with quantum mechanical calculations that predict the vibrational frequencies and intensities for H2O·HO. These calculations gave a binding energy of 5.69 kcal mol-1 for the complex. O2 Formation in Irradiated Ice -- A new mechanism for O2 formation in irradiated ice is presented. This mechanism draws on experimental evidence in the literature to explain the observations of solid O2 on or near the surface of the icy Galilean satellites, Europa and Ganymede. It is proposed that on these bodies, hydrogen peroxide, formed from the radiolysis and photolysis of the ice, is present in highly localized aggregates that hinder O2 diffusion out of the icy surface into the tenuous atmosphere. Further radiolysis and photolysis of these hydrogen peroxide aggregates can then lead to O2 formation via the formation of a short lived water-oxygen atom complex, H2O·O. The O atoms of a pair of these complexes then react rapidly to form O2

Tropospheric oxidation modulates pollution chemistry and greenhouse gas lifetimes. The hydroxyl radical (OH) is the primary oxidant and the main sink for methane, the second-most influential anthropogenic contributor to climate change. OH is produced following the photolysis of ozone, an oxidant, respiratory irritant and greenhouse gas. Trends in methane or ozone are frequently attributed to their sources, but sink-driven variability is less often considered. I investigate the influence of fluctuations in turbulent loss to the Earth’s surface, also known as deposition, on tropospheric ozone concentrations and chemistry over the relatively polluted eastern United States. I use idealized sensitivity simulations with the global chemistry-climate model AM3 to demonstrate that coherent shifts in deposition, on the order recently observed at a long-term measurement site, affect surface ozone concentrations as much as decreases in its precursor emissions have over the past decade. I conclude that a sub-regional deposition measurement network is needed to confidently attribute trends in tropospheric ozone. Next, I turn to the remote marine troposphere to evaluate two theoretical proxies for variability in the methane sink, OH, with observations from the NASA Atmospheric Tomography (ATom) aircraft campaign. The low concentration and short lifetime of OH preclude the development of a representative measurement network to track its fluctuations in space and time. This dearth of constraints has led to discrepancies in the methane lifetime across models that project atmospheric composition and climate. Observational and modeling studies suggest that few processes control OH fluctuations in relatively clean air masses, and the short OH lifetime implies that it is at steady-state (total production is equal to loss). I leverage this chemistry by evaluating a convolution of OH drivers, OH production scaled by the lifetime of OH against its sink with carbon monoxide, as a potential “steady-state” proxy. I also assess the predictive skill of formaldehyde (HCHO), an intermediate product of the methane and OH reaction. I find that both proxies broadly reflect OH on sub-hemispheric scales (2 km altitude by 20° zonal bins) relative to existing, well-mixed proxies that capture, at best, hemispheric OH variability. HCHO is produced following methane loss by reaction with OH and reflects the insolation influence on OH, while the steady-state proxy demonstrates a stronger relationship with OH and offers insight into its sensitivity to a wider array of drivers. Few components—water vapor, nitric oxide, and the photolysis rate of ozone to singlet-d atomic oxygen—dominate steady-state proxy variance in most regions of the remote troposphere, with water vapor controlling the largest spatial extent. Current satellite instruments measure water vapor directly, and other retrievals like nitrogen dioxide columns or aerosol optical depth or could be used to infer nitric acid or the rate of ozone photolysis. Thus satellite observations may be used to derive a steady-state proxy product to infer OH variability and sensitivity in the near-term. HCHO is also retrieved from satellite instruments, and an OH product using satellite-observed HCHO columns is already in development. The relatively high fluctuation frequency of HCHO or the steady-state proxy advances our insight into the connection between OH and its drivers. The observed steady-state proxy demonstrates a widespread sensitivity to water vapor along the ATom flight tracks, and I conclude that an improved and consistent representation of the water vapor distribution is a necessary step in constraining the methane lifetime across global chemistry-climate models.