No description available.

This thesis describes an investigation of the folding and stability of a series of derivatives of the proteins lysozyme and α-lactalbumin which lack one or more of their four native disulphide bridges. Removal of the disulphide bridge which links the N- and C-termini from hen lysozyme results in a three-disulphide derivative (CM^6,127-lysozyme). This has a profound effect on its stability against thermal denaturation, the T_m for unfolding being reduced by 25°C at pH 3.8. Calorimetric measurements performed on this three-disulphide derivative indicate that this reduction in stability may be attributed entirely to an increase in the entropy difference between the native and denatured states. Kinetic refolding studies of CM^6,127-lysozyme using stopped flow optical methods and hydrogen exchange pulse labelling in conjunction with NMR and electrospray ionisation mass spectrometry (ESI-MS) suggest that this reduced stability manifests itself primarily in the α-domain of the protein. A transient intermediate populated during refolding of the unmodified protein can no longer be detected during folding of the derivative resulting in highly cooperative folding under the conditions investigated. The structure and stability of a three- and two-disulphide derivative of the homologous protein, α-lactalbumin have been investigated by NMR spectroscopy. The three-disulphide species, like its lysozyme counterpart, can adopt native structure but this is much more unstable than the intact protein. Removal of a second disulphide bridge, however, destabilises α-lactalbumin to the extent that the native state is no longer formed. Instead, in the presence of Ca²⁺ and high concentrations of salt, a partially structured state is induced which has some elements of tertiary structure present. Novel techniques of ESI-MS have been developed to study protein folding and stability using hydrogen exchange techniques. Applications to the investigation of cooperativity in protein folding, stability in native, partially folded and unfolded states, and the interactions of a partially folded protein with the chaperone GroEL are described.

Upon starvation, individual amoebae of D. discoideum cooperate to produce a cell aggregate. The aggregate then elongates into a slug which moves chemotactically towards food sources. Culmination of the developmental cycle results in the formation of a fruiting body containing differentiated stalk and spore cells. Ammonia is produced naturally in the aggregates during starvation and is implicated in the regulation of the developmental cycle. Addition of excess ammonia leads to inhibition of aggregation, increased slug migration, delay of culmination and the promotion of spore formation. This thesis examines the possible intracellular mechanism(s) through which ammonia might affect developmental processes. It has been postulated that ammonia might act by directly inhibiting a particular enzymatic activity, alternatively it could act as a weak base to raise the pH of the cytosol or an intracellular compartment. To investigate these possibilities, the effects of a variety of weak bases were tested on aggregation and culmination. All of the weak bases tested inhibited development in a dose-dependent way, which argues against the notion that ammonia acts by direct enzyme inhibition since the bases are structurally distinct. In addition, the inhibition potencies of the bases varied according to their protonophoric activity: the most effective protonophores were also the most effective developmental inhibitors. This suggested that the bases were likely to be acting to neutralize an intracellular compartment in these experiments,a result which implicates acidic compartments in the control of development. That weak bases can indeed dissipate the pH of intracellular vesicles was confirmed in vitro by monitoring acidification of isolated vesicles in the presence of the bases. In vivo experiments using ³¹P-NMR probes of the vesicular system confirmed this and showed that cytosolic pH was stable in the presence of bases. Genetic evidence for the role of acidic compartments in development was also gathered. Both the acidification-defective (Hgr) mutants and a mutant created by antisense expression of a fraction of the B subunit cDNA of the vacuolar H⁺- ATPase were defective in development, displaying developmental behaviour reminiscent of weak base-treated wild-type strains. This shows that defective vacuolar acidification can mimic the effect of addition of ammonia and indicates that an acidic vesicle is the target of ammonia In vivo.

No description available.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1978. / Bibliography: leaves 160-164. / Microfiche. / xiv, 164 leaves ill

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1976. / Bibliography: leaves [312]-320. / Microfiche. / xxx, 320 leaves ill

Thesis (Ph. D.)--University of Adelaide, 1986. / Includes bibliographical references.

Thesis (Ph. D.)--McMaster University (Canada), 1990. / Source: Dissertation Abstracts International, Volume: 62-13, Section: A, page: 0000.

Thesis (Ph.D.)--University of Melbourne, Dept. of Biochemistry and Molecular Biology, 2006. / Includes bibliographical references (p. 339-350).

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1990. / Vita. Abstract. Includes bibliographical references (leaves 207-213). Also available via the Internet