Presented in this dissertation are studies on the gas-phase structural features of peptides and peptide fragment ions using mass spectrometry (MS), hydrogen/deuterium (H/D) exchange, infrared multiphoton dissociation (IRMPD) spectroscopy, and computational modeling. Additional studies are presented on the mechanism of hydrogen/deuterium exchange using a model amino acid system. The application of chemical cross-linking to investigate the interaction between two proteins, LexA and RecA, is also presented. Gas-phase structural features can be probed using a number of techniques, and several of the studies presented in this dissertation involve the use of gas-phase H/D exchange. Although the basic mechanism for exchange has been determined, the factors that affect the rate and extent of exchange are not well understood. A computational modeling study of the exchange behavior of asparagine and its methyl ester demonstrated that exchange will occur preferentially at sites of more similar basicity. The distinctive exchange behavior of a model histidine-containing pentapeptide, HAAAA, prompted further studies into the structural features that result in five fast exchanging hydrogens and one slower exchange. Peptide analogues were used to identify the sites of exchange, and IRMPD spectroscopy combined with computational modeling indicated that exchange may occur because interaction with water at those sites results in lower energy structures compared to the other sites. Structural studies were also performed to determine whether the b₂⁺ ion from HAAAA is an oxazolone or diketopiperazine. Although the IRMPD spectrum matched that of a diketopiperazine, H/D exchange and fragmentation studies revealed the presence of both a diketopiperazine and oxazolone structure. Protein-protein interactions perform a vital role in regulating cellular processes. Despite extensive mutational analysis, the binding interaction between LexA and RecA, two proteins involved in the SOS response, is unclear. Chemical cross-linking experiments were undertaken to help target future mutational studies, and these studies identified two possible interactions. The first potential binding interaction is located in the cleft of RecA, and the second interaction may be caused by a LexA dimer binding across the RecA helical groove. The presence of two different binding interactions suggests that LexA may have redundant binding modes for RecA interaction.

A recent shift towards proteomics has seen many structural genomics initiatives set up for high-throughput structure determination using traditional methods of x-ray crystallography and NMR. The next step in the proteomic revolution focuses on the interplay of multi-protein complexes and transient protein-protein interactions, which are involved in many cellular functions. Greater understanding of protein-protein interactions will inevitably lead to better comprehension of the regulation of cellular process, which has implications in biomedical sciences and biotechnology. Even though many high-resolution initiatives focus on proteins and protein complexes, their structure-determination success rates are still low. An emerging approach uses chemical cross-linking and mass spectrometry to derive a set of sparse distance constrains, which can be used for building models of proteins and to map out residues in protein interaction interface based on partial structural information. This technique allows low-resolution identification of protein structures and their interactions in cases where traditional structure determination techniques did not produce results. Chemical cross-linkers have been successfully used for many years in identifying interacting proteins. However, recent advances in mass spectrometry have allowed the identification of exact insertion points of low-abundance cross-links and hence has opened up a new perspective on the use of cross-linkers in combination with computational structure prediction. For protein interaction studies, the approach uses chemical cross-linking information with molecular docking, so that the cross-links are treated as explicit constraints in the calculations. This study focuses on a low-cost and rapid approach to structure prediction, where partial structural information and distance constraints can be used to obtain the relative orientation of interacting proteins and domains, specifically as a rescue strategy where traditional high resolution structure determination methods were unsuccessful. This hybrid biochemical/bioinformatics approach was applied in the determination of structure of the latexin:carboxypeptidase A complex, and succeeded in achieving 4 Å rmsd compared to the crystal structure determined subsequently (Mouradov et al., 2006). Application of the bioinformatics/biochemical approach to multi-domain proteins was carried out on murine acyl-CoA thioesterase 7 (Acot7). X-ray crystallography provided structures of the two separate domains of Acot7, however the full length protein did not crystalise. Combining chemical cross-linking, mass spectrometry, molecular docking and homology modeling we were able to reconstruct how the two domains are arranged in the full length protein (Forwood et al., 2007). Limitations of this technique caused by the enormous complexity of the cross-linking reaction mixtures were identified and emphasized by analysing a large (four protein) complex of DNA polymerase III, where only one inter-protein cross-link was identified. A rapid and cost-effective method for identification of cross-linked peptides using a commercially available cross-linker was developed as part of the overall aim of streamlining the hybrid biochemical/bioinformatics in order for it to become a generally applicable technique for rapid protein structure characterisation (King et al., 2008). Finally an in-house software package was developed for assignment of cross-linked peptides based on m/z values.

A recent shift towards proteomics has seen many structural genomics initiatives set up for high-throughput structure determination using traditional methods of x-ray crystallography and NMR. The next step in the proteomic revolution focuses on the interplay of multi-protein complexes and transient protein-protein interactions, which are involved in many cellular functions. Greater understanding of protein-protein interactions will inevitably lead to better comprehension of the regulation of cellular process, which has implications in biomedical sciences and biotechnology. Even though many high-resolution initiatives focus on proteins and protein complexes, their structure-determination success rates are still low. An emerging approach uses chemical cross-linking and mass spectrometry to derive a set of sparse distance constrains, which can be used for building models of proteins and to map out residues in protein interaction interface based on partial structural information. This technique allows low-resolution identification of protein structures and their interactions in cases where traditional structure determination techniques did not produce results. Chemical cross-linkers have been successfully used for many years in identifying interacting proteins. However, recent advances in mass spectrometry have allowed the identification of exact insertion points of low-abundance cross-links and hence has opened up a new perspective on the use of cross-linkers in combination with computational structure prediction. For protein interaction studies, the approach uses chemical cross-linking information with molecular docking, so that the cross-links are treated as explicit constraints in the calculations. This study focuses on a low-cost and rapid approach to structure prediction, where partial structural information and distance constraints can be used to obtain the relative orientation of interacting proteins and domains, specifically as a rescue strategy where traditional high resolution structure determination methods were unsuccessful. This hybrid biochemical/bioinformatics approach was applied in the determination of structure of the latexin:carboxypeptidase A complex, and succeeded in achieving 4 Å rmsd compared to the crystal structure determined subsequently (Mouradov et al., 2006). Application of the bioinformatics/biochemical approach to multi-domain proteins was carried out on murine acyl-CoA thioesterase 7 (Acot7). X-ray crystallography provided structures of the two separate domains of Acot7, however the full length protein did not crystalise. Combining chemical cross-linking, mass spectrometry, molecular docking and homology modeling we were able to reconstruct how the two domains are arranged in the full length protein (Forwood et al., 2007). Limitations of this technique caused by the enormous complexity of the cross-linking reaction mixtures were identified and emphasized by analysing a large (four protein) complex of DNA polymerase III, where only one inter-protein cross-link was identified. A rapid and cost-effective method for identification of cross-linked peptides using a commercially available cross-linker was developed as part of the overall aim of streamlining the hybrid biochemical/bioinformatics in order for it to become a generally applicable technique for rapid protein structure characterisation (King et al., 2008). Finally an in-house software package was developed for assignment of cross-linked peptides based on m/z values.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D. in Toxicology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 128-138).

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 129-137.

abstract: Treatment of cerebral aneurysms using non-invasive methods has existed for decades. Since the advent of modern endovascular techniques, advancements to embolic materials have largely focused on improving platinum coil technology. However, the recent development of Onyx®, a liquid-delivery precipitating polymer system, has opened the door for a new class of embolic materials--liquid-fill systems. These liquid-fill materials have the potential to provide better treatment outcomes than platinum coils. Initial clinical use of Onyx has proven promising, but not without substantial drawbacks, such as co-delivery of angiotoxic compounds and an extremely technical delivery procedure. This work focuses on formulation, characterization and testing of a novel liquid-to-solid gelling polymer system, based on poly(propylene glycol) diacrylate (PPODA) and pentaerythritol tetrakis(3-mercaptopropionate) (QT). The PPODA-QT system bypasses difficulties associated with Onyx embolization, yet still maintains non-invasive liquid delivery--exhibiting the properties of an ideal embolic material for cerebral aneurysm embolization. To allow for material visibility during clinical delivery, an embolic material must be radio-opaque. The PPODA-QT system was formulated with commercially available contrast agents and the gelling kinetics were studied, as a complete understanding of the gelling process is vital for clinical use. These PPODA-QT formulations underwent in vitro characterization of material properties including cytotoxicity, swelling, and degradation behaviors. Formulation and characterization tests led to an optimized PPODA-QT formulation that was used in subsequent in vivo testing. PPODA-QT formulated with the liquid contrast agent ConrayTM was used in the first in vivo studies. These studies employed a swine aneurysm model to assess initial biocompatibility and test different delivery strategies of PPODA-QT. Results showed good biocompatibility and a suitable delivery strategy, providing justification for further in vivo testing. PPODA-QT was then used in a small scale pilot study to gauge long-term effectiveness of the material in a clinically-relevant aneurysm model. Results from the pilot study showed that PPODA-QT has the capability to provide successful, long-term treatment of model aneurysms as well as facilitate aneurysm healing. / Dissertation/Thesis / Ph.D. Bioengineering 2011

Doctor of Philosophy / Department of Biological and Agricultural Engineering / Donghai Wang / The depletion of petroleum feedstock along with significant concerns about health and environment lead to an interest in alternative green products. Soy protein (SP) adhesives have great potential as a renewable material for wood industries. The obstacle of applying SP-based adhesives is its relatively low water resistance. The overall objective was to enhance the water resistance of SP adhesives through protein and lignin interaction. An improvement of adhesion performance, flowability, and thermal properties of SP adhesives was achieved through the protein and lignin interaction and formation of protein and lignin copolymer. pH adjustment is a simple process to change protein folding and lignin properties. Cleavage of β-O-4 linkage was observed at pH 8.5 and pH 12, resulting in an increase in lignin active groups and the changes in lignin particle size and thermal properties. Cross-linking of protein with lignin took place via carbonyl, amino, and hydroxyl groups. Multiple-point and non-specific interactions between lignin and protein resulted in stronger lignin-protein networks and changes in properties, which improved wet adhesion strength of protein adhesives. In addition, lignin was depolymerized by laccase enzyme with the presence of mediator, TEMPO, to induce a formation of the strong lignin-protein network. The formation of the strong lignin-protein network increased the wet adhesion strength by 106% and the partial wood failure was observed after the three-layer wood test. A better performance was also observed on the three-cycle soaking test. The adhesion performance of SP adhesives was also greatly affected by lignin particle size and the protein to lignin ratio at pH 4.5. The wet adhesion strength of SP adhesives increased as lignin particle size decreased. The protein-lignin adhesive with protein to lignin ratio of 10:2 (w/w) at 12% solid content had the lowest contact angle and the highest wet adhesion strength of 4.66 MPa, which is 53.3% higher than that of 10% pure SP adhesive. Lignin-protein interactions, water resistance property, and glue line pattern had strong influences on an adhesion performance. Lignin and soy protein were modified at pH 4.5, 8.5 and 12. The maximum increase (620%) in water resistance was found at pH 12 with an addition of lignin. After the protein was unfolded (pH 8.5) and denatured (pH 12), it was refolded by shifting pH to 4.5. The better-wet adhesion performance was obtained at pH 4.5, 8.5-4.5 and 12 with rigid glue line. Shifting pH from 8.5 to 4.5 promoted lignin-protein interaction and increased adhesion performance. The protein-lignin adhesives using absolutely renewable materials and practical processes showed an excellent potential to replace the petroleum-based adhesives and fulfill the global demand for green products and technologies.

Doctor of Philosophy / Department of Chemical Engineering / Mary E. Rezac / Catalytic membrane reactors are a class of reactors that utilize a membrane to selectively deliver reactants to catalysts integrated in the membrane. The focus of this research has been on developing and characterizing polymeric catalytic membranes for three-phase hydrogenation reactions, where the membrane functions as a gas/liquid phase contactor allowing selective delivery of hydrogen through the membrane to reach catalytic sites located on the liquid side of the membrane. The benefit of conducting three-phase reactions in this manner is that delivering hydrogen through the membrane to reach catalytic sites avoids the necessity of hydrogen dissolution and diffusion in the liquid phase, which are both inherently low and often described as causing mass-transfer and reaction rate limitations for the reactive system. This work examines two types of membrane reactor systems, porous polytetrafluoroethylene and asymmetric Matrimid membranes, respectively, for the ruthenium catalyzed aqueous phase hydrogenation of levulinic acid. The highly hydrophobic PTFE material provides an almost impermeable barrier to the liquid phase while allowing hydrogen gas to freely transport through the pores to reach catalytic sites located at the liquid/membrane interface. Catalytic rates as a function of hydrogen pressure over the range 0.07 to 5.6 bar are presented and shown to be higher than those of a packed bed reactor under similar reaction conditions. An increasing catalytic benefit was obtained operating at temperatures up to 90 °C, which is attributed to increased hydrogen permeability and avoidance of the decreasing solubility of hydrogen in water with increasing temperature. The membrane reactor was shown to be stable with no decrease in catalytic activity over 200 hours of operation. The Matrimid membrane reactor work demonstrates the feasibility of applying an integrally-skinned asymmetric membrane for an aqueous phase hydrogenation reaction and focuses on the impact that membrane hydrogen permeance and catalyst loading have on catalytic activity. The non-porous nature of the separating layer in the Matrimid membrane allowed successful operation up to 150 °C. The overall catalytic rates were approximately an order of magnitude lower than those achieved in the PTFE membrane reactor system due primarily to significantly lower hydrogen permeances, nevertheless rates were still higher than control experiments. This work also focuses on characterizing Matrimid/solvent thermodynamic relationships for a variety of organic solvents, looking at sorption, diffusion, and polymer relaxation behavior in thin films ranging from 0.1 to 2.0 µm in thickness using quartz crystal microbalance techniques. Diffusion coefficients at infinite dilution for water and C1-C6 alcohols are given as a function of van der Waals molar volume and a clear dependency is shown ranging from 2E-11 to 6.5E-13 cm²/s for water and hexanol, respectively, for 0.26 µm thick films. Diffusion coefficients for all studied vapor penetrants displayed a marked dependence on thickness spanning approximately two orders of magnitude for each respective vapor penetrant over the range 0.1 to 1.0 µm. Chemically cross-linking Matrimid is a method to mitigate some of the relatively high sorption and swelling behavior exhibited in the presence of sorbing species. An in-depth analysis on the vapor phase ethylenediamine cross-linking of Matrimid films and its impact on diffusion, sorption, and relaxation is also described.

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