published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

The structural complexity of glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) and their numerous biological roles, brings forth the need to develop new methods, capable of studying GAGs and their interactions with peptides and proteins under native settings. This thesis explores the development of chemical tools to study heparin/HS binding interactions under physiologically relevant conditions using fluorescence. In chapter 2, we designed peptide-based quinolinium probes to study the structural requirements of cationic peptides required for high affinity peptide-heparin interactions. These fluorescent probes enabled the study of peptide-heparin interactions at nM concentrations allowing the calculation of peptide-heparin binding constants. It was observed that peptides with positive charge displayed on one face of an α-helix in a continuous arrangement bound to heparin with the highest affinity and that heparin likely prefers to bind to these peptides while remaining in an extended conformation. In chapter 3, we set out to study an important biological role of HS which involves the binding and sequestering of proteins at the cell surface, facilitating endocytosis. HS has been implicated in the mechanism of cell penetrating peptide (CPP) cell uptake, with different CPPs showing different degrees of HS dependence on uptake as well as different mechanisms of entry. The role of HS in the mechanism of CPP uptake was investigated in chapter 3 using fluorescent peptide-based probes incorporating fluorophore/quencher pairs. These were used to identify and characterize the ability of heparin/HS to bind and cluster with CPPs to form colloidally stable aggregates. It was shown that the CPP Antp formed much more stable clusters with heparin than the TAT peptide despite both peptides having similar binding affinity for a single heparin chain. These findings were used to explain the cell surface HS dependence of Antp on cell uptake via endocytosis in contrast to the low dependance of TAT on HS and its uptake via translocation. A general model relating the ability of a CPP to cluster surface HS to its preferred mechanism of cell entry was proposed. In chapter 4, a strategy to selectively, and site specifically acylate carbohydrate binding proteins was developed using thioester-based affinity conjugates. It was possible to label maltose binding protein, a periplasmic protein, with high yield and selectivity at a single lysine residue proximal to the maltose binding site. Selective protein labeling could be carried out in bacterial cell extracts and in live bacterial cells. This strategy can potentially be applied to develop protein-based carbohydrate biosensors as well as profile carbohydrate binding proteins in biological samples.

The structural complexity of glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) and their numerous biological roles, brings forth the need to develop new methods, capable of studying GAGs and their interactions with peptides and proteins under native settings. This thesis explores the development of chemical tools to study heparin/HS binding interactions under physiologically relevant conditions using fluorescence. In chapter 2, we designed peptide-based quinolinium probes to study the structural requirements of cationic peptides required for high affinity peptide-heparin interactions. These fluorescent probes enabled the study of peptide-heparin interactions at nM concentrations allowing the calculation of peptide-heparin binding constants. It was observed that peptides with positive charge displayed on one face of an α-helix in a continuous arrangement bound to heparin with the highest affinity and that heparin likely prefers to bind to these peptides while remaining in an extended conformation. In chapter 3, we set out to study an important biological role of HS which involves the binding and sequestering of proteins at the cell surface, facilitating endocytosis. HS has been implicated in the mechanism of cell penetrating peptide (CPP) cell uptake, with different CPPs showing different degrees of HS dependence on uptake as well as different mechanisms of entry. The role of HS in the mechanism of CPP uptake was investigated in chapter 3 using fluorescent peptide-based probes incorporating fluorophore/quencher pairs. These were used to identify and characterize the ability of heparin/HS to bind and cluster with CPPs to form colloidally stable aggregates. It was shown that the CPP Antp formed much more stable clusters with heparin than the TAT peptide despite both peptides having similar binding affinity for a single heparin chain. These findings were used to explain the cell surface HS dependence of Antp on cell uptake via endocytosis in contrast to the low dependance of TAT on HS and its uptake via translocation. A general model relating the ability of a CPP to cluster surface HS to its preferred mechanism of cell entry was proposed. In chapter 4, a strategy to selectively, and site specifically acylate carbohydrate binding proteins was developed using thioester-based affinity conjugates. It was possible to label maltose binding protein, a periplasmic protein, with high yield and selectivity at a single lysine residue proximal to the maltose binding site. Selective protein labeling could be carried out in bacterial cell extracts and in live bacterial cells. This strategy can potentially be applied to develop protein-based carbohydrate biosensors as well as profile carbohydrate binding proteins in biological samples.

One third of proteins in nature have been revealed as metalloproteins, whereas most of them remain uncharacterized, probably due to the lack of robust methods especially for tracking metalloproteins within the living context. Fluorescent labeling is capable to detect biomolecules with molecular resolution in living cells. Tracking metal-binding proteins in living cells by fluorescence could provide invaluable information in understanding their localization and potential functions in the native environment. A synthetic molecular probe NTA-AC was designed and synthesized to track metal-associated proteins in living cells upon chelation with metal ions. The fluorescent probe consists of a small molecular fluorophores, a metal-chelating moiety to direct the metal-chelated probe to the protein targets, and a photo-active crosslinker. Metal being chelated could help further explore potential binding targets and direct the fluorescent agent to the appropriate region, then subsequently covalent linkage to targets could be generated through photo-activation. NTA-AC was therefore chelated with different metals to examine its binding preference to different proteins. The Ni2+-chelating probe was applied to track Ni2+-binding proteins as an example to validate its applicability. Ni2+-NTA-AC preferentially binds to histidine-rich peptides and proteins thus verified its binding specificity. The Ni2+-chelated probe was further exploited to light up over-expressed histidine-rich proteins in Escherichia coli cells to validate its membrane permeability and binding specificity. In addition, the probe was applied to label His-tagged proteins expressed in tobacco plant cells to further evaluate its applicability in detecting and localizing the protein targets in eukaryotic cells. Afterwards, Ni2+-NTA-AC was exploited to track Ni2+-binding proteins in living Helicobacter pylori cells and incorporated with gel electrophoresis and mass spectrometry for protein identification. Many proteins identified are correlated to Ni2+-association and thus validating the applicability of the probe. Bi3+-chelated NTA-AC was therefore used to mine potential targets in H. pylori. Intense fluorescence was observed within H. pylori cells thus indicating the effectiveness of the fluorescent labeling. Protein separation and identification was therefore initiated to trace potential targets, while finding that some of the Bi 3+-coordinated proteins participate in various functioning pathways of the pathogens. The effects of colloidal bismuth subcitrate (CBS) on pH buffering and redox defense systems were therefore determined and verified, confirming that respective proteins could be potential therapeutic targets of the drug. Cr3+-NTA-AC was further applied to human Hep G2 cell line to determine Cr3+-binding targets in mammalian cells. Their localization on mitochondria was revealed, implying the potential effects of Cr3+ on mitochondria. Further confirmation of protein targets was performed through protein separation and identification. Proteins identified could be positively correlated to mitochondrial functions and thus revealing that Cr3+ might exert its effect at mitochondria. Addition of Cr3+ to Hep G2 could prevent mitochondrial fragmentation induced by hyperglycemia, which thus suggests the possible therapeutic function of Cr3+. The extensive application of NTA-AC in tracking Ni2+-, Bi3+- and Cr3+-associated proteins has validated the effectiveness of such strategy in detecting and localizing metalloproteins within the living context and thus could be extended to investigate other metalloproteomes. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Bibliography: leaves 177-181. / v, 181 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 1999

Thesis (Ph. D.)--University of Adelaide, Dept. of Chemistry, 1999. / Includes bibliographical references (leaves 177-181).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references. Also available in print.

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published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy