Fourier transform infrared (FTIR) spectroscopy is a powerful tool for probing protein structure-function relationships. With the use of isotope editing, it can also be employed to elucidate protein-nucleic acid interactions. This technique was used to study the sequence of heat-induced unfolding of the uniformly labeled 13C regulatory subunit (RSU) of E. coli aspartate transcarbamylase (ATCase) with its inhibitor CTP. The absorption of CTP in the amide I' region limits our ability to detect protein conformational changes upon binding of CTP. Therefore, by labeling the protein with 13C shifts the amide I' band ~ 40 cm -1 and clearly separates the protein bands from those of CTP. Variable-temperature (VT) FTIR spectroscopy was then employed to monitor the thermal unfolding of the labeled RSU in the presence and absence of CTP. / In addition, isotope editing was further explored to probe domain-domain interactions of the two domains of RSU using intein technology. Intein technology provides a novel means by which isotope editing can be performed to extract information on protein inter-domain and inter-subunit interactions by spectroscopic analysis but has not yet been exploited in Fourier transform infrared (FTIR) spectroscopy. The objective of this project is to present for the first time the feasibility of segmental labeling through intein-mediated protein ligation (IPL) for the purpose of studying conformational changes by FTIR spectroscopy, using ATCase as a model enzyme. In the first phase of this project, the RSU of ATCase, which houses a Zn-binding domain and a nucleotide binding domain, was reconstructed from its isolated domains using commercially available intein-base expression vectors. As steps towards obtaining an isotope labeled RSU, we have fused each domain to separate inteins. Following affinity purification, the intein tags were chemically cleaved and the reactive ends of the two RSU domains were ligated together to form a peptide. Although ligation was successful, improved yields are required for the FTIR spectroscopic studies.

Defatted soybean was subjected to isolation procedures as follows: NaOH extraction/acid precipitation (NaOH-IE); water extraction/acid precipitation (H2O-IE); NaOH extraction/cryoprecipitation (NaOH-CP) and citric acid extraction/cryoprecipitation (CA-CP). NaOH-CP and CA-CP each resulted in precipitation of one protein mainly consisting of glycinin and beta-conglycinin, respectively. The proteins were subjected to polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, size exclusion-chromatography (SEC) and differential scanning calorimetry (DSC) for characterization. PAGE, SDS-PAGE, SEC and DSC confirmed the homogeneity of NaOH-CP. The gelation properties and structural characteristics of NaOH-CP were investigated; the minimum protein concentration required for formation of a thermally-induced gel from NaOH-CP was 8%. With cold-set gelation, in the presence of 30 mM CaCl2, the minimum protein concentration required for formation of a self-supporting gel was 4%. At 100 mM CaCl 2, cold-set gelation of 10% protein concentration gave gel of strength 7.4 N compared to the 7.8 N gel strength obtained with thermally-induced gel at 16% protein concentration. The effects of heating on the NaOH-CP structure, in the presence of varying concentrations of NaCl and reducing agents, were examined by Fourier transform infrared (FTIR) spectroscopy and DSC. FTIR spectroscopy revealed that at 25°C the NaOH-CP protein showed four bands at 1633, 1650, 1667, and 1689 cm-1. At 85°C, the temperature associated with gelation, the four bands decreased in intensity with the simultaneous increase in the intensity of a band at 1644 cm-1 as result of the protein unfolding, furthermore two new bands at 1616 and 1685 cm -1 appeared. The latter two bands are attributed to intermolecular mu-sheet formed during gelation. The NaOH-CP did not exhibit any aggregation band between 25--100°C in the presence of NaCl. The DSC studies revealed that the protein denatured at approximately 102°

Polymeric depressant adsorption on the surface of talc was investigated using in situ Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy. The polymeric depressants investigated were an unmodified polyacrylamide (Polymer-N) and a hydroxyl substituted polyacrylamide (Polymer-H), an unmodified dextrin (Dextrin WY) and a hydroxylpropyl substituted dextrin (HP Dextrin), and carboxymethyl cellulose (CMC). The main objectives of this study were to evaluate the thermodynamic properties of adsorption, the kinetics of adsorption and desorption, and the mechanisms driving adsorption. / Thesis (PhDApSc(MineralsandMaterials))--University of South Australia, 2007.

Thin coatings of photoresponsive, pyrimidine-terminated molecules, attached to gold or quartz substrates in contact with water, undego photodimerisation and wettability changes when irradiated with UV light at 280 and 240mm. Spin-casting and chemisorption techniques were used to prepare the thin films. / Thesis (PhDApSc)--University of South Australia, 2000.

Thin coatings of photoresponsive, pyrimidine-terminated molecules, attached to gold or quartz substrates in contact with water, undego photodimerisation and wettability changes when irradiated with UV light at 280 and 240mm. Spin-casting and chemisorption techniques were used to prepare the thin films. / Thesis (PhDApSc)--University of South Australia, 2000.

Thesis (Ph.D.). / Written for the Dept. of Food Science and Agricultural Chemistry, Macdonald College of McGill University. Title from title page of PDF (viewed 2008/08/04). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Food Science and Agricultural Chemistry, Macdonald College of McGill University. Title from title page of PDF (viewed 2008/08/04). Includes bibliographical references.

Thesis (Ph. D.)--Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Dr Amyn.S.Teja; Committee Member: Dr Haskell Beckham; Committee Member: Dr Peter. J. Ludovice; Committee Member: Dr William.J.Koros; Committee Member: Dr. Thomas H. Sanders.

Thesis (Ph. D.)--University of Wyoming, 2007. / Title from PDF title page (viewed on Feb. 6, 2009). Includes bibliographical references.

Thesis (Ph.D.)--Drexel University, 2009. / Includes abstract and vita. Includes bibliographical references (leaves 259-273).