In systems biology, material balances, kinetic models, and thermodynamic boundary conditions are increasingly used for metabolic network analysis. It is remarkable that the reversibility of enzyme-catalyzed reactions and the influence of cytosolic conditions are often neglected in kinetic models. In fact, enzyme-catalyzed reactions in numerous metabolic pathways such as in glycolysis are often reversible, i.e., they only proceed until an equilibrium state is reached and not until the substrate is completely consumed. Here, we propose the use of irreversible thermodynamics to describe the kinetic approximation to the equilibrium state in a consistent way with very few adjustable parameters. Using a flux-force approach allowed describing the influence of cytosolic conditions on the kinetics by only one single parameter. The approach was applied to reaction steps 2 and 9 of glycolysis (i.e., the phosphoglucose isomerase reaction from glucose 6-phosphate to fructose 6-phosphate and the enolase-catalyzed reaction from 2-phosphoglycerate to phosphoenolpyruvate and water). The temperature dependence of the kinetic parameter fulfills the Arrhenius relation and the derived activation energies are plausible. All the data obtained in this work were measured efficiently and accurately by means of isothermal titration calorimetry (ITC). The combination of calorimetric monitoring with simple flux-force relations has the potential for adequate consideration of cytosolic conditions in a simple manner.

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

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The exploitation of the relationship between the chemical and physical properties of materials is the hallmark of advancing science throughout the world. The basic understanding of how and why molecules react and interact with each other in different environments allows for the discovery and implementation of new materials and devices that not only advance the state of human life but continually change the planet. The work described in this dissertation generally falls under three diverse categories: functionalization of fullerenes, investigation of host/guest interactions in solution, and the synthesis and characterization of ion containing polymers. The separation and functionalization of fullerenes is a recent and exciting area of research. The separation methods outlined are intended to increase the availability of endohedral metallofullerenes by decreasing their cost of production. Functionalized fullerene species were achieved through Bingel and Prato reactions to provide materials with novel functional groups. These materials may be further utilized in photovoltaic or other organic electronic devices. The characterization of noncovalent interactions between different molecules in solution is the focus of supramolecular chemistry. Isothermal Titration Calorimetry stands out as one of the best, among the many methods used to elucidate the characteristics of these systems. The binding of bis- imidazolium and paraquat guests with macrocyclic host molecules has been explored in this work. The measurements of the association constants for these systems will aid in the ongoing synthesis of new host/guest systems. Ion containing polymers were synthesized and characterized for their use in electroactive devices. Imidazolium containing polymers with bulky anions were synthesized on low glass transition polymer chains. These materials had enhanced ion conductivity and may eventually be used in electronic actuator materials. / Ph. D.

Advanced analytical techniques were utilized to investigate the intermolecular forces in several nanostructured materials. Techniques including, but not limited to, isothermal titration calorimetry (ITC), variable temperature Fourier transform infrared (FTIR) spectroscopy, and ultraviolet-visible (UV-Vis) thermal curves were used to study the fundamental interactions present in various nanomaterials, and to further probe the influence of these interactions on the overall behavior of the material. The areas of focus included self-assembly of surfactant micelles, polycation complexation of DNA, and temperature-dependent hydrogen bonding in polymeric systems. ITC was successfully used to determine the low critical micelle concentration (CMC) for a novel gemini surfactant with limited water solubility. CMCs were measured at decreasing methanol molar fractions (xMeOH) in water and the resulting linear relationship between CMC and methanol concentration was used to mathematically extrapolate to a predicted CMC at xMeOH = 0. Using this technique, the CMC value for the novel gemini surfactant was predicted to be 0.037 ± 0.004 mM. This extrapolation technique was also validated with surfactant standards. ITC was also used to investigate the binding thermodynamics of polyplex formation with polycations and DNA. The imidazolium-containing and trehalose-based polycations were both found to have endothermic, entropically driven binding with DNA, while the adenine-containing polycation exhibited exothermic DNA binding. In addition, ITC was also used to confirm the stoichiometric binding ratio of linear polyethylenimine and DNA polyplexes as determined by a novel NMR method. Dynamic light scattering (DLS) and zeta potential measurements were also performed to determine the size and surface charge of polyplexes. Circular dichroism (CD) and FTIR spectroscopies provided information regarding the structural changes that may occur in the DNA upon complexation with polymers. UV-Vis thermal curves indicated that polyplexes exhibit a greater thermal stability than DNA by itself. Variable temperature FTIR spectroscopy was used to quantitatively compare the hydrogen bonding behavior of multi-walled carbon nanotube (MWCNT)-polyurethane composites. Spectra were collected from 35 to 185 deg C for samples containing various weight percent loadings of MWCNTs with different hydrogen bonding surface functionalities. Peak fitting analysis was performed in the carbonyl-stretching region for each sample, and the hydrogen-bonding index (Rindex) was reported. Rindex values were used to quantitatively compare all of the composite samples in regards to temperature effects, weight percent loadings of MWCNTs, and the different functionalizations. In general, higher weight percent loadings of the MWCNTs resulted in greater Rindex values and increased hydrogen bond dissociation temperatures. In addition, at 5 and 10 wt% loadings the initial Rindex values displayed a trend that tracked well with the increasing hydrogen bonding capacity of the various surface functionalities. / Ph. D.

<p>The intricate interplay of biomolecules acting together, rather than alone, provides insight into the most basic of cellular functions, such as cell signaling, metabolism, defense, and, ultimately, the creation of life. Inherent in each of these processes is an evolutionary tendency towards increased efficiency by means of biolgocial synergy-- the ability of individual elements of a system to produce a combined effect that is different and often greater than the sum of the effects of the parts. Modern biochemists are challenged to find model systems to characterize biological synergy.</p><p>We discuss the multicomponent, enzyme complex the cellulosome as a model system of biological synergy. Native cellulosomes comprise numerous carbohydrate-active binding proteins and enzymes designed for the efficient degradation of plant cell wall matrix polysaccharides, namely cellulose. Cellulosomes are modular enzyme complexes, comparable to enzyme "legos" that may be readily constructed into multiple geometries by synthetic design. Cellulosomal enzymes provide means to measure protein efficiency with altered complex geometry through assay of enzymatic activity as a function of geometry.</p><p>Cellulosomes are known to be highly efficient at cellulose depolymerization, and current debates on the molecular origins of this efficiency suggest two related effects provide this efficiency: i) substrate targeting, which argues that the localization of the enzyme complex at the interface of insoluble cell wall polysaccharides facilitates substrate depolymerization; and ii) proximity effects, which describe the implicit benefit for co-localizing multiple enzymes with divergent substrate preferences on the activity of the whole complex.</p><p>Substrate targeting can be traced to the activity of a single protein, the cellulosomal scaffoldin cellulose binding module CBM3a that is thought to uniquely bind highly crystalline, insoluble cellulose. We introduce methods to develop a molecular understanding of the substrate preferences for CBM3a on soluble and insoluble cellulosic substrates. Using pivaloylysis of cellulose triacetate, we obtain multiple soluble cello-oligosaccharides with increasing degree of glucose polymerization (DP) from glucose (DP1) to cellodecaose (DP10) in high yield. Using calorimetry and centrifugal titrations, cello-oligosacharides were shown to not bind Clostridial cellulolyticum CMB3a. We developed AFM cantilever functionalization protocols to immobilize CBM3a and then probe the interfacial binding between CBM3a and a cellulose nanocrystal thin film using force spectroscopy. Specific binding at the interface was demonstrated in reference to a control protein that does not bind cellulose. The results indicate that i) CBM3a specifically binds nanocrystalline cellulose and ii) specific interfacial binding may be probed by force spectroscopy with the proper introduction of controls and blocking agents.</p><p>The question of enzyme proximity effects in the cellulosome must be answered by assaying the activity of cellulosomal cellulases in response to cellulosome geometry. The kinetic characterization of cellulases requires robust and reproducible assays to quantify functional cellulase content of from recombinant enzyme preparations. To facilitate the real-time routine assay of cellulase activity, we developed a custom synthesis of a fluorogenic cellulase substrate based on the cellohexaoside of Driguez and co-workers (vide infra). Two routes to synthesize a key thiophenyl glycoside building block were presented, with the more concise route providing the disaccharide in four steps from a commercial starting material. The disaccharide building blocks were coupled by chemical activation to yield the fully protected cellohexaoside over additional six steps. Future work will include the elaboration of this compound into an underivatized FRET-paired hexasaccharide and its subsequent use in cellulase activity assays.</p><p>This dissertation also covers an experimental system for the evaluation of methonium desolvation thermodynamics. Methonium (-N+Me3, Am) is an organic cation widely distributed in biological systems. The appearance of methonium in biological transmitters and receptors seems at odds with the large unfavorable desolvation free energy reported for tetramethylammonium (TMA+), a frequently utilized surrogate of methonium. We report an experimental system that facilitates incremental internalization of methonium within the molecular cavity of cucurbit[7]uril (CB[7]).</p><p>Using a combination of experimental and computational studies we show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by a remarkably small desolvation enthalpy of just 0.5±0.3 kcal*mol-1, a value significantly less endothermic than those values suggested from gas-phase model studies (+49.3 kcal*mol-1). More surprisingly, the incremental withdrawal of methonium surface from water produces a non- monotonic response in desolvation enthalpy. A partially desolvated state exists, in which a portion of the methonium group remains exposed to solvent. This structure incurs an increased enthalpic penalty of ~3 kcal*mol-1 compared to other solvation states. We attribute this observation to the pre- encapsulation de-wetting of the methonium surface. Together, our results offer a rationale for the wide biological distribution of methonium and suggest limitations to computational estimates of binding affinities based on simple parameterization of solvent-accessible surface area.</p> / Dissertation

CD2AP is a member of the CD2AP/CIN85 family of adaptors and involved in several cellular processes, such as kidney podocyte development, actin mediated membrane trafficking and T cell activation. It contains three SH3 domains whose binding properties and interaction partners remain largely unexplored. The CD2AP SH3 interaction with the novel partner Rab5-activating GEF RIN3 was studied extensively by isothermal titration calorimetry (ITC), peptide scanning arrays, mutagenesis and X-ray crystallography. Mapping of the interaction regions showed that human RIN3 contains two binding sites for the CD2AP SH3 domains. From these studies, the CD2AP SH3 recognition motif P-x-P/A-x-x-R emerged. Two crystal structures (1.65 &Aring; and 1.2 &Aring;) of the SH3 1 and SH3-2 domains in complex with RIN3 epitopes 1 and 2 respectively revealed that these residues serve as anchoring points. With the aid of bioinformatics tools, this motif was used to conduct a peptide array-based screen for additional signalling partner candidates. One of the hits was the Arf-GAP ARAP1. ITC data indicate that the three SH3 domains differentially recognise three ARAP1 epitopes, with the first ARAP1 epitope binding to SH3-2 in the nanomolar range. A crystal structure (1.6 &Aring;) of the SH3-2 domain in complex with the first ARAP1 epitope implicates two additional anchoring residues that extend beyond the PPII helical region of the canonical motif. The CD2AP/ARAP1 interaction was confirmed in podocytes and cancer cells at the endogenous protein level. Even though RIN3 and ARAP1 are involved in membrane trafficking, a direct link to CD2AP had not been reported before. Other candidates from the peptide array analyses were also investigated by ITC. In conclusion, this study led to the elucidation of the CD2AP SH3-1 and SH3-2 domain binding signatures and the identification of putative novel binding partners for all three SH3 domains. Lastly, insight was gained into the binding preferences of CD2AP SH3-3.