Amphibians contain a rich chemical arsenal in their skin glands, vital for their defence against predators, both large and small. The peptides secreted by the frogs have a range of biological activities. These include both antibacterial and anticancer activity, others are neuropeptides, while some exhibit antifungal and antimalarial activity. Peptides are usually sequenced using positive ion mass spectrometry ( MS ). However, negative ion MS can also provide valuable sequencing information. Under negative ion MS conditions, the presence of a Cys residue is immediately identified by the facile side chain loss of H [subscript 2] S. The position of the Cys residue is determined by the formation of a side chain induced backbone cleavage ion. When a Cys residue is in the C - terminal position of a peptide, the spectrum is dominated by the loss of both H [subscript 2] S and CO [subscript 2]. Negative ion MS can also be used to identify and sequence intramolecular disulfide bridged peptides. The disulfide bridge is immediately identified by the facile loss of H [subscript 2] S [subscript 2] from the parent anion. Once the disulfide bridge is cleaved, further amide cleavages provide much of the sequence of the peptide, including the residues originally within the disulfide link. When one of the disulfide bridged Cys residues is in the C - terminal position, the major fragmentation is the loss of H [subscript 2] S [subscript 2] and CO [subscipt 2] from the parent ion. The negative ion mass spectra of citropin 1.1 and synthetically modified analogues show an unusual loss of an internal Val residue from the ( M - H ) - parent ion. This rearrangement requires the decomposing anion to have an α - helical structure. The skin secretions of Litoria peronii or Peron ' s Tree Frog contain five novel peptides which have been named peroneins. Four pro - peroneins are present in the summer secretions only. The biologically active peptides caerulein 1.1, caerin 1.1 and caerin 2.1 are also present in the glandular secretions. / Thesis (Ph.D.)--School of Chemistry and Physics, 2006.

Cell penetrating peptides (CPPs) have drawn the attention of researchers due to their ability to internalize large cargos into cells including cancer cells. The mechanism(s) with which the peptides enter the cell, however, is/are not clear and full of controversy. The peptide conformations and their microenvironment in live cells had been unknown until the development of a technique developed in our lab. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm-1, thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals. / On the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and b-strand in the cytoplasm, and possibly assembling as b-sheets in the nucleus. Next, label-free transportan was studied with the same methodology. The peptide, besides predominantly a-helix, adopted a significant portion of b-sheet conformation in the cytoplasm and nucleolus, which is different from the peptide in aqueous solution. The peptide microenvironment was also probed through H-bonding reported by the tyrosine Fermi doublet. Transportan displayed a tendency to accumulate in the cytoplasm over time which was unlike penetratin, which concentrated in the nucleus. The relative concentration of CPPs in various locations of live melanoma cells was directly estimated from the Raman spectra using average Phe concentration in the cell as an internal standard. / The rapid entry and almost uniform cellular distribution of both peptides, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of CPP internalization under the conditions of study was probably non-endocytotic. Last, transportan and penetratin were studied using polarized Raman spectroscopy for more detailed vibrational spectroscopic information of the two peptides in water and TFE solutions. The majority of the bands in the Raman spectra of the peptides were highly polarized, consistent with the high symmetry of aromatic ring side chain vibrational bands dispersed throughout the spectra. This work has provided new insights into the structure of CPPs in live cells and in solutions. / by Jing Ye. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Using 2 - D NMR spectroscopy, the structure of the sex pheromone from Litoria splendida has been determined, in order to elucidate its mode of transport through the aquatic environment. The peptide was found form an α - helical structure, with a central flexible hinge region. The mode of transport through the aquatic environment has been discussed in relation to the structure. Previous work indicated that the Australian amphibian host defence skin peptides that inhibit neuronal nitric oxide synthase ( nNOS ) were likely to act indirectly on the enzyme, by binding to the co - enzyme of nNOS, calmodulin. [superscript 15] N labelled calmodulin was expressed and purified via a bacterial protein expression system and a series of 2 - D NMR [superscript 15] N - HSQC titrations was performed with Australian amphibian host defence skin peptides. in order to determine whether these peptides bind to calmodulin. The three peptides tested were found to bind, and with differing strengths of interaction. One of these was selected for further study. [superscript 15] N and [superscript 13] C doubly labelled calmodulin was then prepared in order to study the complex between this protein and the selected peptide, caerin 1.8, an Australian amphibian skin peptide isolated from Litoria chloris. A series of 3 - D NMR spectra has been recorded on this complex. The backbone atom resonances have been assigned for free calmodulin and for the calmodulin - peptide complex, using a combination of main chain directed and sequential assignment strategies. By analysing the changes in chemical shift that occur upon binding the peptide, it was determined that the mode of binding involves a stronger interaction with the C - terminal domain than the N - terminal domain. / Thesis (Ph.D.)--School of Chemistry and Physics, 2006.

Recombinant human macrophage colony stimulating factor beta (rhm-CSFβ) is a glycoprotein that stimulates the proliferation, differentiation and survival of cells belonging to the monocyte-macrophage lineage. It contains nine inter-subunit and intra-subunit disulfide bonds and represents an excellent model system for studying disulfide bond formation during protein folding because the assembly of its monomeric subunits and the maturation of its biological activity depend on the progressive formation of the correct disulfide structure during in vitro folding. Knowledge obtained from these studies can be potentially useful in understanding the roles of disulfide bond formation during protein folding in general. rhm-CSF8 was modified by partial reduction of disulfide bonds, yielding CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ. The modification did not affect the biological activity, stability, or the overall conformation of the protein. However, the C-terminal regions near the modification sites were shown to exhibit faster deuterium exchange behavior as a result of the chemical modification, indicating that the C-terminal regions became more flexible. Folding kinetics of rhm-CSFβ and CN¹⁵⁷'¹⁵⁹-modified rhm-CSFβ were shown to be essentially the same, suggesting that the modification did not affect the folding kinetics of the oxidized rhm-CSFβ. The denatured and reduced rhm-CSFβ was refolded with the aid of a chemical oxidant. The data indicated that the in vitro folding rhm-CSFβ proceeded via multiple pathways involving monomeric and dimeric intermediates. Disulfide bond shuffling catalyzed by GSH/GSSG represented an important isomerization step in folding. A dimeric intermediate, D-SS8-cam2, was isolated and identified as a kinetic trap, perhaps requiring significant structural arrangement to convert to the native protein. The heterogeneous folding mixture detected by both disulfide bond quenching and H/D pulsed labeling indicate that rhm -CSFβ folding is a diffusion like process as described by the folding funnel model. / Graduation date: 2003

The SUC2 locus of Saccharomyces cerevisiae encodes two forms of invertase; a constitutively expressed cytoplasmic enzyme and a glucose-repressible secreted and glycosylated enzyme which is initially produced with an amino-terminal signal peptide. The coding sequence of the SUC2 locus has been placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere based yeast plasmid vector from which invertase is expressed in a Sue" strain of yeast. Oligonucleotide-directed mutagenesis has been used to create a PstI site in the gene at the point encoding the signal peptide cleavage site. An internal methionine codon, the translation start for the cytoplasmic invertase, has been replaced by a serine codon. Mutants in the signal peptide sequence have been produced by replacing the region of the gene upstream of the PstI site with synthetic oligonucleotide cassettes with mixtures of nucleotides at several positions. The mutants could be divided into three classes based on their ability to secrete invertase. The first class of mutants produced secreted invertase, but in reduced amount. There is no obvious correlation between mutation and phenotype. The second class, represented by mutant 4-55B, also exhibited a reduced level of invertase, but a significant fraction (30%) of the enzyme is intracellular. This mutant had a delay in signal peptide cleavage which retards passage of invertase through the secretory pathway. The third class was defective in secretion. Most were defective in translocation from the cytoplasm to the lumen of the endoplasmic reticulum (ER), and produced enzymatically active, non-glycosylated pre-invertase in the cytoplasm. This class of mutant invertases, when transcribed and translated in vitro, was not processed by canine pancreas signal recognition particle (SRP) and microsomes. Comparison of the sequences of the mutant signal peptides of this non-translocating class identifies amino acids at the extreme amino-terminus as the causative defect. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

In attempts to explore the usefulness of the newly introduced electron capture dissociation (ECD) mass spectrometry for structural analysis of peptides/proteins, different fundamental aspects of the ECD method were investigated using a combination of controlled experiments and high-level theoretical calculations. The relative propensity for dissociation (RPD) of different amino acid residues were extracted from a series of ECD experiments using a common peptide model of RGGGXGGGR, where X was varied systematically among 20 common amino acid residues. Although polar and aromatic amino acid residues were found to behave differently, there exists a fair correlation between the experimental RPD values of aliphatic amino acid residues and the corresponding calculated hydrogen atom affinities of the nearby carbonyl groups. The existence of this correlation reinforces the importance of "hot hydrogen-atom model". From the same set of experiments, the side chain loss reactions of the reduced precursor ions and the zn+• species were extracted. To account for the observed secondary fragments, several generalized dissociation pathways were proposed. The energetics of these dissociation pathways were evaluated theoretically with truncated peptide models using ab initio and DFT calculations; and the kinetics of several competitive reactions were evaluated using Rice-Ramsberger-Kassel-Marcus (RRKM) calculations. / The effect of charge carriers on ECD of peptides/proteins was also studied. Peptides charged through protonation of different basic amino acid residues were found to give ECD spectra of different complexities. The formation of b-/y- and atypical internal fragment ions in peptides with histidines (and lysine, to a lesser extent) as proton carriers was attributed to the higher electron-proton recombination energy as revealed from the energy cycle diagram. Peptides charged through attachment of divalent metal ions were found to give very different ECD spectra. It was believed that typical c/z • fragments were formed from neutralization reactions involving electron-proton recombination; whereas a/b/y fragments were formed from reaction involving electron-metal ion recombination. The preference of recombination channels was somehow related to the electronic configurations of the divalent metal ions. / Fung Yi Man. / "July 2006." / Adviser: T. W. Chan. / Source: Dissertation Abstracts International, Volume: 68-08, Section: B, page: 5254. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 180-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Due to the large gaps in the fossil record, the evolutionary history of the mammalian order Cetacea is incomplete and controversial. Increasingly researchers are utilizing molecular and biochemical procedures to supplement cetacean paleontology. One of these methods is the comparison of amino acid sequences of myoglobin among species of this order. since this method is time-consuming and expensive, an alternative procedure is desirable.

Peptide YY (PYY) is a gut-derived hormone that is renowned for its effects on satiety. Reduced satiety in obese people has been attributed to low fasting and postprandial PYY levels. However, it has not been determined whether low PYY levels are the cause or the outcome of obesity. Moreover, the long-term role of PYY in regulating energy balance is unclear. Results presented in this thesis, using PYY-deficient mice (PYY-/-) and PYY transgenic mice (PYYtg) highlight that PYY indeed has an important role in regulating energy balance and glucose homeostasis in vivo. PYY knockout mice became obese with ageing or high-fat feeding linked to a hyperinsulinemic phenotype associated with hypersecretion of insulin from isolated pancreatic islets. These findings suggested that PYY deficiency may be a predisposing factor for the development of obesity and type 2 diabetes. On the other hand, PYYtg mice exhibited decreased adiposity and increased metabolism under high-fat feeding. Furthermore, PYYtg/ob mice had improved glucose tolerance and decreased adiposity. These latter studies suggested that high circulating PYY levels may protect against the development of obesity and type 2 diabetes. Interestingly, both animal models support PYY as an important regulator of the somatotropic axis. These preliminary findings prompted investigations in understanding whether low PYY levels may be a predisposing factor for the development of obesity and type 2 diabetes in human subjects. In a population of healthy human subjects that had a predisposition to the development of type 2 diabetes and obesity, fasting PYY levels were lower than in normal subjects. Moreover, low fasting PYY levels strongly correlated with decreased insulin sensitivity and high levels of fasting insulin. Collectively, these findings suggest that low circulating levels of PYY could contribute to increased adiposity, insulin resistance and type 2 diabetes. Therefore determination of PYY levels may be a method of detecting whether people are predisposed to becoming obese and insulin resistant. This work also suggests that treatments that enhance circulating PYY levels may be protective in the development of obesity and type 2 diabetes.

Venoms, toxins and host-defence systems constitute rich sources of biologically active molecules, many of which have enormous therapeutic and biotechnological potential. In particular, peptides are often a significant component of these chemical arsenals, and are fundamentally important as biological effector molecules. The research presented in this thesis is centred on the isolation and investigation of peptides from both frogs and spiders, and endeavours to probe the important structural and mechanistic features of these bioactive compounds. The skin peptide profiles of interspecific hybrids between the green tree frog Litoria caerulea and the magnificent tree frog Litoria splendida have been investigated in a ninemonth survey. Fourteen peptides were characterised primarily using mass spectrometry, of which three had not been identified previously in the skin secretions of either parent. A number of these peptides are antibacterial agents, while others effectively inhibit the formation of nitric oxide by neuronal nitric oxide synthase. Implications for the genetics and expression of amphibian dermal peptides are also discussed. The majority of frogs of the genus Litoria contain at least one peptide in their glandular secretion capable of inhibiting the formation of nitric oxide by the enzyme neuronal nitric oxide synthase. This was proposed to occur by preventing the association of the regulatory cofactor, Ca²⁺ -calmodulin, with its binding site on the enzyme. Non-covalent binding of the amphibian peptides to calmodulin in the presence of Ca²⁺ has been confirmed using electrospray ionisation mass spectrometry, by the observation of complexes in the gas phase with a 1 : 1 : 4 calmodulin / peptide / Ca²⁺ stoichiometry. In addition, the structure and binding interactions of caerin 1.8, a potent nitric oxide synthase inhibitor, have been further probed using mass spectrometry and nuclear magnetic resonance spectroscopy techniques. Recently a number of small, disulfide - containing neuropeptides of the signiferin and riparin families have been characterised from the skin secretion of frogs of the Crinia genus. Of these, signiferin 1 and riparin 1.1 are both ten residue peptides with similar primary sequences, however appear to have a significantly different spectrum of bioactivity. Although both act at cholecystokinin-2 receptors, signiferin 1 is smooth muscle active while riparin 1.1 is not, and instead causes proliferation of lymphocytes. The three-dimensional structures of these peptides were determined using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Both signiferin 1 and riparin 1.1 adopt β - turn type conformations, however differences in these structures may be responsible for the variation in biological activity noted for these peptides. The dermal secretions of most Australian frogs contain at least one broad-spectrum peptide antibiotic, and often a series of peptides with differing activity to afford greater protection against microbial pathogens. Solid state nuclear magnetic resonance spectroscopy studies were carried out to investigate the interaction of a number of these antibacterial peptides with anionic model membranes, and the results are compared with work previously reported using neutral lipids. It appears the peptides may have a different mode of interaction with the membranes depending upon the charge of the lipid head group. The cupiennin 1 peptides have been identified in the venom of the neotropical wandering spider, Cupiennius salei, and demonstrate potent wide-spectrum antibacterial activity. Primary sequence analysis of these peptides suggests a unique amphipathic structure distinctly different from that of other potentially helical cationic antimicrobial peptides isolated thus far. Using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations, cupiennin 1a was found to adopt an α- helical structure with a flexible central hinge region in membrane mimicking solvents. Following this, nuclear magnetic resonance spectroscopy methods were used to further probe the antibacterial and the newly identified neuronal nitric oxide synthase inhibitory activity of this peptide. / Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, Discipline of Chemistry, 2006

The skin extracts from amphibians have been investigated for over fifty years and have been found to contain numerous components with therapeutic and medicinal uses. Host - defence compounds are secreted onto the dorsal surface of the animal from specialised granular glands in response to a variety of stimuli, such as stress induced by a predator. Isolated peptides can exhibit either pharmacological properties or antibiotic activity. Previous studies isolated a potent hypotensive neuropeptide, crinia angiotensin II, within skin secretions of the Australian frog Crinia georgiana. This prompted further investigations into the isolation and sequence determination of host - defence compounds from other species in this genus - C. signifera, C. riparia and C. deserticola. Fifteen novel peptides were identified. The major peptide components were potent disulfide containing neuropeptides of a type not observed in other Australian anurans that have been previously investigated. The remaining peptides demonstrate either antibiotic activity or inhibit the enzyme neuronal nitric oxide synthase. The skin components from anurans of the Litoria genus have been extensively studied, with a number of peptides exhibiting both antibacterial and pharmacological activity. The skin secretion of Litoria dentata has been investigated, with five novel peptides identified. The neuropeptide tryptophyllin L 1.3 was previously isolated from the related frog L. rubella. Other components that are unique in structure have not yet been tested for biological activity. The parasitic disease malaria is responsible for over one million deaths per year. The increase in resistance of current antimalarial compounds has led to the development of new treatments from various animal - derived peptide antimicrobials. A number of amphibian peptides and their derivatives were investigated as potential antiplasmodial agents against the malaria parasite Plasmodium falciparum. Results indicate that these compounds inhibit parasite growth with minimal haemolytic activity, making them promising tools for malaria research.The defence chemistry of amphibian neuropeptides has been extensively studied and is important in understanding both the ecology and physiology of the vertebrate. Neuropeptides are classified into groups with similar structural characteristics. Biological activity occurs via interaction with a G protein - coupled receptor. The most studied of all amphibian neuropeptides is caerulein, which has a similar spectrum of activity to the mammalian peptide cholecystokinin. This includes smooth muscle contraction that occurs via interaction with cholecystokinin receptors. The pharmacological activity of Australian anuran neuropeptides from various genera was investigated. Two biological assays were conducted - a smooth muscle contraction test and a lymphocyte proliferation assay. A range of neuropeptides contracted smooth muscle at nanomolar concentrations, while others only proliferated lymphocytes. Some peptides were inactive in both assays. Young marsupials are born at an immature stage of development and rely on immune protection provided by the mother. Eugenin is a host - defence compound isolated from pouch secretions of the Tammar wallaby. The immunomodulator activates CCK2 receptors, resulting in lymphocyte proliferation. Therefore, eugenin stimulates immune cells in the pouch providing vital immune protection for pouch young. / Thesis (Ph.D.)--School of Chemistry and Physics, 2006.