Expanding the Genetic Code to Probe Biological Systems

The chemical modification of proteins has been a longstanding interest in the scientific community. In addition to the natural modifications necessary for life to function, unnatural covalent modifications are particularly useful because they facilitate research efforts that require the precise manipulation of protein, including the installation of fluorescent labels, post-translational modification mimics, and affinity reagents. Historically, appending such modifications onto proteins was achieved by generating covalent adducts onto one of the twenty canonical amino acids. However, such modifications are not site-selective, and may interfere with the native function of the modified protein. Genetic code expansion can overcome the limitations inherent to canonical amino acid modification, especially when bioorthogonal functional groups are incorporated. Using orthogonal aminoacyl-tRNA synthetase-tRNA pairs, one can reliably obtain homogenous samples of modified protein in a site-selective manner. In order to fully understand the steric requirements of a rationally designed pyrrolysyl-tRNA synthetase, several large meta-substituted phenylalanine derivatives were synthesized and incorporated into superfolder green fluorescent protein using this synthetase. All synthesized substrates were incorporated, albeit with differing incorporation efficiencies. Moreover, this synthetase was found to incorporate 3-formyl-phenylalanine, an aldehyde-based amino acid that can be directly installed onto proteins. Prior to this work, only indirect post-translational approaches could install aldehydes, and these methodologies were limited as to where the modification could occur. Aldehyde labeling occurs rapidly at neutral pH, and peptide cyclization has been accomplished using the aldehyde to form a thiazolidine linkage with an N-terminal cysteine, further demonstrating the rich chemistry available to aldehydes. Finally, efforts to optimize azidophenylalanine bioconjugation led to a kinetic investigation of copper-catalyzed click chemistry, which led to a revision of the currently accepted mechanism; it is proposed that copper-catalyzed click chemistry requires two separate copper-chelating events, with one equivalent of copper binding to azide, and another equivalent of copper binding to alkyne. The work presented herein demonstrates that phenylalanine derivatives are useful substrates for probing and manipulating biological systems, as well as providing opportunities for discovery in chemical biology

Methods to identify novel α-helical peptides that bind to LMO4

Protein-protein interactions (PPIs) play an essential role in regulating cells. LIM Domain-Only protein 4 (LMO4) is a transcriptional co-regulatory protein that functions entirely through PPIs. It contributes to developmental processes and the pathogenesis of breast cancer, although the mechanisms are only partially known. The development of inhibitors of LMO4 could form useful research tools for understanding these mechanisms and may form the basis of new breast cancer therapies. Previously, the Matthews Laboratory has developed peptide inhibitors of LMO4 based on natural binding partners (β-strand peptides). The hypotheses is that α-helical peptides can be more specific than β-strand peptides, it should be possible to generate α-helical peptide binders of LMO4 using natural α-helices as templates. The aims were to develop two methods to identify novel α-helical peptides that bind to LMO4: a split EGFP complementation (spEGFP) system providing a high through-put method for the initial screening of a library of α-helical peptides; and, a Yeast Two Hybrid Competition Assay (Y2HCA) as an orthogonal method to validate hits and provide an assessment of their relative binding affinities. For the Y2HCA, a new construct was added to create a series of constructs that relatively assess affinities of weakly binding peptides. The spEGFP system was expanded, introducing controls and a construct to screen a library of α-helical peptides for affinity to LMO4LIM1. Naturally occurring α-helices were considered and tested as potential templates. Chemical transformation and DNA extraction protocols for BL-21(DE3) cells were developed to enable efficient library expression and screening. This thesis provides mechanisms to identify α-helical peptides that bind to LMO4 and the ground work for a high-throughput library screen

Novel routes to defined post translational modifications using non-canonical amino acids.

Proteins are inherently limited by the properties of their constituent amino acids and attempt to overcome this by using post translational modifications (PTMs). PTMs are highly specific and can effectively modulate protein function faster than simple up or down regulation of protein production. However, PTMs often require a suite of other proteins to regulate and perform the modification to ensure accuracy, which can be hard to engineer into synthetic proteins. By introducing new chemistry into proteins via noncanonical amino acids (ncAAs) we can expand the range of new non-native PTMs that we can explore. Non-native PTMs (nnPTMs), have the potential to be both bioorthogonal and easily transferable between proteins. This thesis examines the effects of engineering nnPTMs into superfolder Green Fluorescent Protein (sfGFP) to study the effects on fluorescence of: 1) modification with small molecules (Chapter 3), 2) Creation of covalent protein dimers (Chapter4), 3) Interfacing proteins to carbon nanomaterials (Chapter 5), and 4) Look at the effects of engineering cooperativity using ncAAs (Chapter6). Most of this work focused on the ncAA, p-azido-L-phenylalanine (azF) as it has several properties that would be desirable for use in proteins such as photo reactivity and selective reactivity with alkynes. Moreover, as azF can be incorporated into any target protein in a range of hosts, it is an ideal starting point to engineer nnPTMs that are easily transferable. Throughout this thesis the importance of intricate hydrogen bonding networks and water channels, to the function of a protein, is made apparent through a range of in silico, structural and biophysical techniques. In silico modelling is used throughout to predict; the effects of nnPTMs on sfGFP structure (Chapter 3 and Chapter 6), dimer interfaces in Chapter 4, and show functional linking between sfGFP and carbon nanotubes in Chapter 5

Enteroviral evolution: interspecies recombination and implications for picornavirus research

The original aim of the project was to determine whether a non-poliovirus HEV could evolve to use the poliovirus receptor (PVR). A variety of methods were used to exploit aspects of the evolutionary capacity of viruses to achieve this goal. Although the aim was not attained, the investigation of recombinants between different HEVs yielded interesting results, which were pursued further. Two approaches were developed: in vitro generation of recombinant viruses and phenotypic analysis of such chimeras and the selection for recombinant viruses in vitro. In vitro generation of reciprocal recombinants between the structural and the non-structural coding region of coxsackievirus A21 (CVA21) and poliovirus type 3 (PV3) was initiated. Transfected and passaged chimeras did not produce infectious virions. Immunofluorescence analysis of VP1 protein expression suggested that the recombinants were not acytopathic. A series of assays were then carried out to investigate the nature of the defect. HeLa S10 translation/transcription reactions of the in vitro generated recombinants expressed the correct protein-processing pattern suggesting efficient processing occurred in vitro. Replication assays demonstrated that the chimeras were replication competent. Trans-encapsidation experiments were then carried out and preliminary results strongly suggested that the defect could lie at the packaging level. Selection of recombinants in vivo, without predetermining the crossover sites, was also conducted. Under the conditions used, recombinant between CVA21 and PV3 impaired genomes and echovirus 7 (EV7) and PV3 impaired genomes proved to be unsuccessful. Characterisation of the impaired parental genomes used for the experiment needs to be carried out. However, recent reports of recombinants between Sabin polioviruses and HEV-C confirm the possibility of such a recombination event occurring and emphasize concerns regarding the success of the polio eradication program

Functional characterization of Mucin-Associated Surface Protein (MASP) in the human parasite Trypanosoma cruzi

MASPs are members of a multigenic family recently identified during the sequencing of the T. cruzi CL Brener genome. This family contains around 1,400 members, consisting of approximately 6% of the whole coding genes. Highly conserved N- and C-terminal domains, which encode a signal peptide and GPI-anchor addition site respectively, and a hypervariable central region, characterize MASPs. Members of this family are predominantly expressed in the infective trypomastigote form. We hypothesized that members of the T. cruzi MASP protein family play a major role in the interaction of the parasite with the host cell. In order to investigate a putative role for T. cruzi MASP at the host-pathogen interface, we have used MASP as a bait protein against the human proteome using a high-throughput platform that we have recently established for identifying protein-protein interactions between pathogens and theirs hosts. Yeast two-hybrid screens identified human SNAPIN as one of two major MASP interacting proteins. SNAPIN is a member of the SNARE protein complex, which may have a role in a calcium-dependent exocytosis. The MASP-SNAPIN interaction was further validated using in vivo co-Affinity Purification and in vitro pull-down assays. Immunofluorescence assays showed human SNAPIN is recruited to the parasite surface during invasion. Co-localization experiments indicated that SNAPIN is associated with the late endosomes and lysosomes. Supporting our initial hypothesis, SNAPIN depletion using siRNA oligomers in HeLa cells and snapin-/- in Mouse Embryonic Fibroblast (MEF) cells significantly inhibited T. cruzi invasion, suggesting a role for SNAPIN in this process. Lysosomes in snapin-/- MEF cells displayed aberrant morphology and distribution and the parasites did not recruit host lysosomes efficiently when compared to wild-type cells. This was likely due to an impaired calcium-dependent lysosome exocytosis in snapin-/- MEF cells. SNAPIN was translocated to the plasma membrane upon calcium influx induced by a calcium ionophore (Ionomycin), resulting in the exposure of the luminal domain of SNAPIN to the extracelluar space. Leishmania tarentolae transgenic strains expressing two different MASP proteins were shown to trigger intracellular calcium transients in HeLa cells, presumably by injuring the cell membrane. We propose that T. cruzi MASP plays a role in wounding the plasma membrane of the host cell, which in turn elicits a transient intracellular calcium flux and leads to the translocation of lysosome-associated SNAPIN to the plasma membrane. Human SNAPIN, through its exposed luminal domain would then provide an anchor for the entry to the parasite into the cell. The mechanism of T. cruzi MASP evoked calcium influx in the host cell membrane remains under investigation

The effect of post translational modification and oligomerisation on the structure-function relationship of horseradish and fluorescent proteins.

Protein function is inherently linked to amino acid sequence; however, this vocabulary is innately limited. One natural method to supplement protein chemistry is the application of PTM. This modification has significant implications for both the structure and function of protein and can now be emulated with non-natural chemistry. This thesis examines the influence PTM has, on protein stability (Chapter 3), function (Chapter 4), structure (Chapter 5), and how nnPTM can be applied to induce functional communication (Chapters 6 and 7). This thesis first explores the expression of HRP in E. coli (Chapter 3), wherein it is demonstrated that soluble HRP expression, is achieved by the inclusion of the full-length proto-HRP sequence. These proto-HRP regions are then shown to cleave, and additionally, it is also shown that the removal of unoccupied N-linked glycosylation sites from the surface of HRP decreases the rate of precipitation observed in recombinant HRP. Next, recombinant apo-HRP is investigated as a corrective agent in a commercial immunoassay (Chapter 4), to establish if the corrective function of glycosylated apo-HRP can be achieved without glycosylation. Evidence generated indicates that glycosylation of apo-HRP is essential for the removal of false-positive rogue signalling. Subsequently, Chapter 5 explores the influence of PTM on HRP’s structural rigidity. Using fluorescence emission to assess the red edge excitation shift of W117, it is observed that both haem binding and glycosylation increase protein structural rigidity, and this is confirmed by CD spectra. The latter two chapters of this thesis explore the potential of chromophore communication which can be induced by nnPTM. Together, both in silico interface modelling, and nnAA incorporation, is utilised for the formation of two fluorescent dimers. Firstly, this system was applied to the structurally similar combination of sfGFP and mCherry (Chapter 6). In which the enhanced chromophore proximity resulted in a significant decrease in function of mCherry, contrary to expectation. In Chapter 7 sfGFP and cytochrome b562 were dimerised to generate dimeric protein which could transfer energy by way of FRET (Chapter 7). Lastly, both nnPTM and natural PTM were combined in the formation of trimeric protein which was linked by SPAAC and disulphide linkage

Targeting infectious species by Structure-guided Fragment-based Drug Discovery and in silico approach

We are living in a post-antibiotic era in which several human pathogens have developed multidrug resistance and few new antibiotics are being discovered. In addition to the challenge of the emergence of antibiotic resistance, the bacterial population also harbours mechanisms that increase pathogenicity and virulence by tolerating antibiotics and avoiding the host immune system. The emergence of species with multidrug resistance (MDR) and extensively drug resistant species (XDR) has alerted us to the danger of infection threats. For instance, Mycobacterium abscessus (Mab) is a rapidly growing multidrug-resistant species of nontuberculous mycobacteria (NTM) in addition to the well-characterised highly virulent species of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter subspecies) reported by the Centers for Disease Control and Prevention (CDC) and the World Health Organisation (WHO). There is therefore an urgent need to develop novel classes of antibiotics against these pathogens. In addition to resistance being problematic, antibiotic tolerance also adds another level of complexity during the process of treatment by compromising non-essential activities or creating dormant ‘persister’ states. The mechanisms of triggering persister phenotypes seem rather unclear but Toxin-antitoxin (TA) complexes have been reported to play a significant role in triggering persister phenotype. In this thesis, novel and validated targets are exploited using fragment-based drug discovery (FBDD) and in silico approaches to design unconventional antibiotics that could extend the scope of current treatment options and reverse some of the effects of living in a post-antibiotic era. Firstly, three biological targets that work as enzymes (FtsZ, MurB and CoaD) were investigated by cloning, purification, and crystallisation with the aim of solving structures of apo states or complexes with molecules in addition to fragment screening using one or more biophysical methods to identify suitable fragment complexes for structural characterization. The Second part of the thesis concerns toxin-antitoxin (TA) complexes, however most experiments focused on antitoxin components to avoid any possible binding of fragments or compounds to the active site of the toxin that might hinder its activity. Lastly, peptidomimetics were designed and screened in silico to find how this could build on the knowledge gained from previous experiments and improve on current strategies for targeting toxin-antitoxin complexes