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Protein-nucleic acid interactions play essential roles in all living cells in various cellular functions. The study of these interactions can reveal important structural and functional information. UV cross-linking of nucleic acids to proteins in combination with mass spectrometry is a powerful technique to identify proteins, peptides and the amino acids involved in intermolecular interactions within nucleic acid-protein complexes. However, the mass spectrometric identification of cross-linked nucleic acid-protein heteroconjugates in complex mixtures and MS/MS characterisation of the specific sites of cross-linking is a challenging task. In this investigation, novel tools and methods have been developed for the investigation of DNA-protein interactions using UV cross-linking and mass spectrometry. These tools were developed towards their application for the characterisation of the complex between the eukaryotic DNA repair protein Tyrosyl-DNA phosphodiesterase 1 (Tdp1) and its DNA substrates. DNA-Tdp1 UV cross-linking was optimised using purified recombinant human Tdp1 and radioactively labelled DNA oligonucleotides containing UV photoactivatable 4- thio-thymidine or 5-iodouracil. Tdp1-DNA heteroconjugates were detected by SDS PAGE and Phosphorimaging. In order to analyse the DNA-Tdp1 heteroconjugates by mass spectrometry, they must first be enriched and hydrolysed by a protease and a nuclease. Here, a novel sample preparation protocol was developed for the enrichment of Tdp1 oligonucleotide-peptide heteroconjugates. Detection and analysis of oligonucleotide-peptide heteroconjugates using mass spectrometry is a challenging task. As a tool to optimise the various parameters involved, a synthetic DNA oligonucleotide-peptide heteroconjugate was constructed using click chemistry. RP-HPLC/ESI-FT-ICR-MS on a Bruker 12T SolariX in conjunction with CID fragmentation was used to unambiguously identify the site of the cross-link. Lastly, a novel 18O labeling approach was introduced to facilitate the identification of DNA-protein cross-links. This approach was shown to be suitable for the labeling of heteroconjugate species by testing it with the click heteroconjugate.

The objective of this study was to improve canola protein gelation properties with the use of enzymes. Both cross-linking and limited proteolysis were explored. Enzyme treatments were performed prior to heat induced gelation. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. Enzymatic cross-linking with transglutaminase was shown to improve the gelation of canola protein isolate (CPI). To the contrary, proteolysis with trypsin, ficin and bromelin, did not enhance the gelation properties of CPI. / October 2006

One of the main problems in healthcare is the loss or failure of organs or tissues resulting from diseases, post-surgery complications, trauma or organ failure. As a result of tissue and organ shortage, there is a need for biomaterials designed to promote tissue regeneration resulting in good quality repair of tissues or organs, to maintain or repair biological function. Collagen, as one of the main proteins in the human body, has been extensively used in the development of biomaterials which can be used as tissue substitutes or can assist in tissue regeneration. Before commercialisation is allowed all biomaterials must prove to be functional and suitable for clinical use. Therefore, the evaluation of biomaterials requires rigorous and relevant testing. Biomaterials must be able to perform with an appropriate host response in a specific application. Tests must provide information to understand the host response, long-term outcome and issues pertaining to these. In the research reported in this thesis, an acellular porcine derived cross-linked collagen-based biomaterial (Permacol surgical implant) was analysed with a wide range of evaluation and compared to acellular noncross-linked and cellular, naturally cross-linked, equivalents. These matrices were characterized relating to their structure, composition and mechanical and biochemical properties. In addition, biological characterisation was performed through several studies designed to evaluate and compare biological responses in vitro, as well as in situ assessment of biocompatibility and effectiveness as a repair material and as bulking tissue. Permacol surgical implant was shown to be biocompatible, effective and efficient when used as bulking tissue and for soft tissue repair; furthermore, this biomaterial was resistant to enzymatic digestion and tolerant to bacterial presence suggesting that it could be used in some complicated clinical situations.

Formaldehyde cross-linking has been used to study protein-protein interactions in cells. Its short spacer arm, ability to permeate through cell membrane and the reversibility of the cross-linking reaction makes this a desirable cross-linker for in vivo studies. Although it has been widely used as a cross-linking reagent, the detailed chemistry behind protein cross-linking is not well understood. In vitro studies conducted under extended incubation periods (2 days) have shown that a multitude of amino acids are reactive to formaldehyde and that residue accessibility appears to play a role in reactivity. How applicable these findings are to formaldehyde cross-linking studies done under in vivo conditions (10-20 min incubations) is unclear. The chemistry of formaldehyde cross-linking was therefore investigated in model peptides under conditions similar to those used in in vivo studies. It was observed that only a subset of amino acids (amino termini and side chains of lysine and tryptophan) that were found reactive under extended incubation times was reactive in the much shorter incubation period. No cross-linking was detected between peptides, and elevating the peptide and formaldehyde concentrations resulted in only a minimal amount of cross-linked peptides. The relationship between residue accessibility and formaldehyde reactivity was assessed in model proteins that contain a more complex tertiary structure. It was shown that the extent of formaldehyde reactivity was dependent on the state of protein unfolding, i.e., solvent accessibility of reactive residues, and that an unfolded protein showed a significantly higher number of formaldehyde-induced modifications than a folded form, with lysine being the predominant reactive site. Formaldehyde treatment of proteins in their native form resulted in a low number of modifications even under an increased incubation time, suggesting that the protein remains folded during the course of the reaction. This is important for in vivo cross-linking studies where specificity and stability of protein-protein interactions is dictated by protein tertiary structure.

The objective of this study was to improve canola protein gelation properties with the use of enzymes. Both cross-linking and limited proteolysis were explored. Enzyme treatments were performed prior to heat induced gelation. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. Enzymatic cross-linking with transglutaminase was shown to improve the gelation of canola protein isolate (CPI). To the contrary, proteolysis with trypsin, ficin and bromelin, did not enhance the gelation properties of CPI.

The objective of this study was to improve canola protein gelation properties with the use of enzymes. Both cross-linking and limited proteolysis were explored. Enzyme treatments were performed prior to heat induced gelation. A texture analyzer, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy were used to characterize the resulting networks. Enzymatic cross-linking with transglutaminase was shown to improve the gelation of canola protein isolate (CPI). To the contrary, proteolysis with trypsin, ficin and bromelin, did not enhance the gelation properties of CPI.

Formaldehyde cross-linking has been used to study protein-protein interactions in cells. Its short spacer arm, ability to permeate through cell membrane and the reversibility of the cross-linking reaction makes this a desirable cross-linker for in vivo studies. Although it has been widely used as a cross-linking reagent, the detailed chemistry behind protein cross-linking is not well understood. In vitro studies conducted under extended incubation periods (2 days) have shown that a multitude of amino acids are reactive to formaldehyde and that residue accessibility appears to play a role in reactivity. How applicable these findings are to formaldehyde cross-linking studies done under in vivo conditions (10-20 min incubations) is unclear. The chemistry of formaldehyde cross-linking was therefore investigated in model peptides under conditions similar to those used in in vivo studies. It was observed that only a subset of amino acids (amino termini and side chains of lysine and tryptophan) that were found reactive under extended incubation times was reactive in the much shorter incubation period. No cross-linking was detected between peptides, and elevating the peptide and formaldehyde concentrations resulted in only a minimal amount of cross-linked peptides. The relationship between residue accessibility and formaldehyde reactivity was assessed in model proteins that contain a more complex tertiary structure. It was shown that the extent of formaldehyde reactivity was dependent on the state of protein unfolding, i.e., solvent accessibility of reactive residues, and that an unfolded protein showed a significantly higher number of formaldehyde-induced modifications than a folded form, with lysine being the predominant reactive site. Formaldehyde treatment of proteins in their native form resulted in a low number of modifications even under an increased incubation time, suggesting that the protein remains folded during the course of the reaction. This is important for in vivo cross-linking studies where specificity and stability of protein-protein interactions is dictated by protein tertiary structure.

abstract: In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment. / Dissertation/Thesis / Ph.D. Chemistry 2011