Although integral membrane proteins represent nearly a quarter of the genes present in both prokaryotes and eukaryotes, progress in this area of research is often hindered due to the nature of their hydrophobic environment. Elucidating the folding pathway of these proteins is essential to understand many membrane mediated biological processes such as signal transduction, ion transport and chemotaxis. The wealth of structural and genetic information on bacteriorhodpsin renders it an ideal model system for the study of membrane proteins. Detailed studies however, necessitate efficient methods for its overexpression and purification. Previous expression systems have reported difficulty in obtaining good yields and simple purification procedures. This thesis investigates a variety of alternative expression and purification systems for the bacterio-opsin gene in Escherichia coli. With sufficient protein, site directed mutagenesis is performed to mutate three proline residues present in the membranous region of bacteriorhodopsin to alanine. The folding kinetics of these mutants is investigated using stopped flow fluorimetry to determine whether proline isomerisation is responsible for a slow step in the folding pathway of bacteriorhodopsin. Comparison of the results with those of the folding kinetics of wild type showed proline isomerisation not to be responsible for the slow step in the pathway. More recent studies have suggested that the slow step may be due to refolding conditions and lateral pressure the lipids impose upon the protein as well as pH. Separate structural studies using mass spectrometry aimed to study the rates of isotopic exchange of amide and side chain protons in bacteriorhodopsin. Low resolution results obtained using matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) prompted the investigation of electrospray ionisation mass spectrometry (ESI-MS). Techniques for sample preparation were optimised by investigating a variety of solvent systems and initial deuteration experiments performed.

Thesis (Ph. D.)--University of Washington. / Includes bibliographical references.

Thesis (Ph.D.)--Bowling Green State University, 2008. / Document formatted into pages; contains xii, 116 p. : ill. Includes bibliographical references.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Physics and Mathematical Physics, 2001. / Includes bibliographical references (leaves 174-188). Also available electronically.

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The work described in this thesis was performed at the Physics Department, Rhodes University during 1958 and 1959 under the supervision of Professor J.A. Gledhill. Use was made of a vacuum ultra-violet spectrograph which had been constructed in the Physics Department (1.1) and modifications to be described were made to this instrument. The instrument was used for studying the effects of oxygen on the fluorescence excitation spectrum of Anthracene.

The use of fluorescent molecules as probes of protein conformation is recognized as a technique which provides very specific information and has been applied, in recent years, to the study of the role of tropomyosin (TM) in the regulation of contractile processes. The isolation and sequencing of TM from horse blood platelets (P-TM) has shown it to be different from muscle TM, especially near the NH₂-and COOH-termini. These differences have been suggested to weaken end-to-end interaction of P-TM molecules. TM's are two chain coiled coils and P-TM has cysteine residues at the penultimate COOH-terminus position of adjacent chains. These can be labelled with sulfhydryl-specific fluorescent probes that reflect conformational changes in that region of the molecule via changes in their emission characteristics. The results of experiments on both pyrene (Py) (40) and acrylodan (AD) labelled P-TM show that there is a preferred interaction of the COOH-terminus of P-TM with the NH₂-terminus of cardiac TM over that with the NH₂-terminus of P-TM. This indicates that the altered NH₂-terminus of P-TM, with respect to muscle TM, is responsible for the relative loss of polymerizability of P-TM at low salt concentration. Addition of actin to the Py-P-TM (40) and AD-P-TM species showed changes in emission characteristics indicative of binding to the F-actin filaments, suggesting that the presence of the probes had not affected the function of the P-TM adversely. However, the presence of pyrenes at the COOH-terminus seemed to reduce further the ability of P-TM to self-polymerize. Thermal denaturation of AD-P-TM, AD-C-TM and AD-labelled truncated P-TM followed by fluorescence polarization suggested that, contrary to the theory of Skolnick and Holtzer on the stability of two chain coiled coils, the region towards the COOH-terminus is among the last to lose its helical character. / Science, Faculty of / Chemistry, Department of / Graduate

For many years there has been a great interest among the scientific community in the study of the hydroxyl radical, HO. This interest stems from the fundamental role played by this molecule in the photochemistry of the atmosphere, mainly as a cleansing agent of environmental pollutants. Knowing the concentration of the radical would enable scientists to corroborate current atmospheric models and to predict future trends in the atmosphere. Even though there is a great interest in the determination of atmospheric concentrations of this molecule, the task has been very difficult. This is mainly due to the lack of a method sensitive enough to detect concentrations around 106 molecules per cubic centimeter. The most accurate method presently available is the method of laser induced fluorescence using the fluorescence assay with gas expansion technique (LIF-FAGE). This method involves low pressure excitation of HO from its ground state to its lowest electronic excited state and observing the consequent fluorescence around 309 nm. The procedure is done at a pressure of 5 Torr to maximize the fluorescence lifetime of the radical and to minimize the interference of photolytic species. Background determination is achieved by chemical modulation using isobutane in a second channel of the same cell which removes the HO signal. In this study an assessment of the level of ozone interference in LIFF AGE has been done by calculating the relative population distribution of HO among its rotational levels and from this, determining its temperature. When the laser passes through the excitation detection cell it photolyses the ozone present producing in this way the highly reactive 0 1(D). When this molecule reacts with water or with isobutane it produces HO, and this is the source of interference in the actual measurements. In the determination of the relative population distributions of the different HO species, it was found that the naturally occurring HO has a thermal distribution with a temperature of about 300 K. The HO molecules produced from the reaction of 0 1(D) with isobutane also showed a thermal distribution with a temperature of about 230 K. On the other hand, the HO produced from the reaction of 0 1(D) with water did not show a thermal distribution. Two distinct temperatures were observed for this case: one around 200 K for values of K = 1 to 4, and the second one around 3000 K for values of K = 5 to 6. These values agree with previous experimental results for LIF methods by other authors except for the fact that the deviation from the first temperature determined by other authors starts at K = 6 or 7.

Lam Wai-yean. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1978. / Bibliography: leaves 108-109.

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