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As polyethylene (PE) products become more prevalent in a wide variety of engineering applications, it is critical to understand their chemical and mechanical degradation to prevent premature failure. Sunlight radiation has been recognized as the most severe environmental factor causing chemical degradation in PE. Furthermore, PE is also susceptible to mechanical failure through creep rupture and stress cracking. This dissertation focuses on the impact of these degradation mechanisms on the lifetime of PE. PE products designed for sunlight exposure are required to be stabilized with antioxidant (AO) to retard polymer degradation. In this dissertation, photochemical depletion of AO in PE samples exposed to artificial sunlight was monitored by the oxidative induction time (OIT) testing. A mathematical model was developed by implementing the photochemical reaction scheme, radiation attenuation, and diffusion of AO and oxygen. The model successfully described the AO depletion profiles and obtained a relationship between radiation intensity and AO depletion rate, based on which an equation was proposed to predict the AO depletion in PE in exposure to natural sunlight. Mechanical degradation of PE was studied by investigating creep rupture and stress cracking in thin-wall PE tubes. A test setup was designed to apply static and cyclic internal pressures to thin-wall PE tubes and to record their failure times. The pressure tests were accelerated using elevated temperatures. The failure time (and number of cycles) was predicted using two shifting methods for the dry-cooling applications. Furthermore, this dissertation investigated wave formation of PE geomembranes undergoing thermal expansion during installation. Finite element method (FEM) was used to simulate the formation of waves and quantify the tensile strains along the deformed geomembrane. The model was validated by previous experimental studies. Additionally, the effect of overburden pressure on waves was studied.

F1-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by α3β3. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E. coli F1-ATPase by hydrogen-deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F1 complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F1-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get "stuck" (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional "greasy bearing" model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F1-ATPase is attributed to the absence of a c10 ring that stabilizes the rotation axis in intact FoF1. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors.

Proteins are involved in virtually every biochemical process. A comprehensive characterization of factors that govern protein function is essential for understanding the biomedical aspects of human health. This dissertation aims to develop complementary mass spectrometry-based methods and apply them to solve problems pertaining to the area of protein structure, folding and dynamics. ‎Chapter 1 uses fast photochemical oxidation of proteins (FPOP) to characterize partially disordered conformers populated under semi-denaturing conditions. In FPOP, ·OH generated by laser photolysis of H2O2 introduces oxidative modifications at solvent accessible side chains. By contrast, buried sites are protected from radical attack. Using apomyoglobin (aMb), it was demonstrated that under optimized conditions undesired can be almost completely eliminated and detailed structural information can be obtained. ‎Chapter 3 combines FPOP with submillisecond mixing to enable studying early events in protein folding. aMb served as a model system for these measurements. Spatially-resolved changes in solvent accessibility follow the folding process. Data revealed that early aMb folding events are driven by both local and sequence-remote docking of hydrophobic side chains. Assembly of a partially formed scaffold after 0.2 ms is followed by stepwise consolidation that ultimately yields the native state. The submillisecond mixer used improved the time resolution by a factor of 50 compared to earlier FPOP experiments. Submillisecond mixing in conjunction with slower mixing techniques help monitor completes folding pathways, from fractions of a millisecond all the way to minutes. ‎Chapter 4 uses ion mobility mass spectrometry (IM-MS) to explore the structural relationship between semi folded solution and gas phase protein conformers. Collision cross sections (CCSs) provide a measure of analyte size. Mb was used as model system because it follows a sequential unfolding pathway that comprises two partially disordered states. IM-MS data showed that the degree of gas phase unfolding is not strongly correlated with the corresponding solution. Gas phase unfolding as well as collapse events can lead to disparities between gaseous and solution structures for partially unfolded proteins. IM-MS data on non-native conformers should therefore be interpreted with caution. ‎Chapter 5 uses HDX-MS to examine the role of conformational dynamics for the function of multi-protein molecular machines such as FoF1 ATP synthase. HDX-MS monitors backbone deuteration kinetics in the presence of D2O. Disordered segments exchange more rapidly than those in tightly folded regions. Measurements of spatially-resolved deuterium are performed using LC-MS. It was found that the H-bonding network of key power transmission elements is insensitive to PMF-induced mechanical stress. Unexpectedly, HDX-MS reveals a pronounced destabilization of the g C-terminus during rotational catalysis under PMF. The behavior of g is attributed to kinetic friction within the apical rotor bearing.