Suzana Straus


Research Classification

Research Interests

Antimicrobial peptides
Protein-protein interactions
Viral membrane proteins

Relevant Thesis-Based Degree Programs

Affiliations to Research Centres, Institutes & Clusters


Research Methodology

Nuclear magnetic resonance (NMR)
Other biophysical techniques (e.g. CD, ITC, fluorescence)


Master's students
Doctoral students
Any time / year round
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I am open to hosting Visiting International Research Students (non-degree, up to 12 months).

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Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Defining the role of partially covalent weak interactions in chemical reactions (2022)

Weak interactions, commonly known as secondary bond interactions (SBIs), have received far less attention than their stronger counterparts (i.e., primary bonds, e.g., ionic, metallic, and covalent bonds). The most mainstream perspective is that those interactions are induced by the attraction between partially positive and partially negative areas on molecular surfaces. This electrostatics-driven model has been widely accepted as it is straightforward, intuitive, and widely applicable; however, more and more computational and experimental discoveries have thrown this “simple and practical” theory into question. Other quantum chemical effects like charge transfer cannot be ignored if a generalized picture of weak interactions is required. Due to the relatively weak bond strengths, it is often difficult to study weak interactions exclusively and accurately. With synchrotron X-ray absorption spectroscopy (XAS) in combination with different computational methods, the nature of two common weak interactions (i.e., halogen bonding XB and chalcogen bonding ChB) has been comprehensively investigated. To begin with, two types of model systems (i.e., hypervalent halogen salts and bis-triazole-pyridinium series) were chosen to study the degree of charge transfer in XB and ChB. These studies have given evidence for a substantial degree of covalent contribution in both XB and ChB interactions. The relevance of such interactions in modulating chemical processes has also been explored. For example, the significance of the charge transfer component in XB was confirmed in a catalyzed Ritter-like process (carbon-halogen bond cleavage). We have further demonstrated the important role of ChB in processes ranging from petroleum-coke modification to pharmaceutical drug synthesis. The obtained data has shown that both XBs and ChBs should be considered as partially covalent interactions and the covalent contributions can be tuned by the properties of involved electron donor/acceptor pairs. Those results are intended for a more rational design of the weak-interaction-assisted systems in many fields, such as catalysis, synthesis, anion recognition, and molecular electronics.

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Mechanism of action of two active antimicrobial peptides derived from aurein 2.2 (2020)

With the increasing problem of antibiotic resistance, coupled with the limited number of newly developed antibiotics, therapeutic alternatives are urgently required. Host defense peptides (HDPs), also known as antimicrobial peptides (AMPs), are promising candidates as they have multifaceted anti-infective properties as well as a lower likelihood of causing antibiotic resistance. HDPs are ubiquitous in nature and constitute an important part of the innate immune system of almost all life forms. However, only a few peptides are in clinical trials due to issues such as systemic toxicity, proteolytic degradation and short half-life from renal/hepatic clearance. Therefore, it is important to minimize the negative impacts while improving the antimicrobial activity at the same time. Part of this thesis describes the various functions of HDPs including antibacterial, antibiofilm, immunomodulatory activities, along with the associated mechanisms of action (MOAs). The determination of antibacterial and antibiofilm MOAs of active HDPs forms the basis of this thesis, with a description of commonly used methods. From an array of aurein 2.2 analogue peptides, peptide 73 and peptide 77 were chosen for further study as they showed the highest antibacterial activity. Furthermore, both analogue peptides demonstrated better antibiofilm activity than aurein 2.2. Different biophysical techniques were used to investigate the structure – function relationship as well as the antibiofilm MOA of these analogue peptides. Specifically, the binding interaction with the alarmone nucleotide ppGpp was investigated, to determine whether the analogues function by inhibiting the bacterial stringent response. In addition, biological techniques were also employed to characterize their in vitro activity against bacteria. Finally, the antibacterial and antibiofilm activities of several HDPs are compared to attempt to assess whether these anti-infective properties are linked or independent and future experiments are proposed. Overall, the analogue peptides demonstrated higher antibacterial and antibiofilm activities compared to aurein 2.2. In addition, both have a slightly different antibiofilm mechanism of action compared to the antibiofilm peptide, IDR-1018. The results from this thesis will help to establish a foundation for future HDP design so that they can be used as therapeutics to combat antimicrobial resistance (AMR).

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Spectroscopic Studies of Halogen Bonding in Model Systems: From one end of the electromagnetic spectrum to the other (2019)

At its simplest, chemical bonding involves a combination of two dominant contributions: direct electrostatics (ionic) and electron sharing (covalent). The relative importance of these contributors has been the subject of signif- icant study in primary (intramolecular) chemical interactions. For example, the relevance and importance of covalent contributions has been a primary focus of transition metal chemistry for decades. For weaker secondary chem- ical interactions such as hydrogen bonding (HB) and halogen bonding (XB), the prevailing view in the literature is that electrostatic interactions are so dominant that covalent contributions are negligible. A notable exception is that of so-called symmetric hydrogen bonds, which exhibit large covalent contributions. With X-ray Absorption Spectroscopy (XAS), we have provided the first direct experimental evidence of covalency in XB. From such studies, we ob- serve that XB exhibit a significantly higher degree of covalency compared with HB counterparts of similar bond strength. Notably, the degree of co- valency in certain XBs is equivalent to that observed in transition metal halides. Our studies provide information of the electronic changes that oc- cur in both the charge donor and charge acceptor in model systems, affording us a unique experimental view of these weak interactions. We also demon- strate the importance of covalent contributions in XBs by showing the effect of covalency in the electron transfer properties in XB-modified dye sensi- tised solar cells. These results lead us to conclude that XBs should more generally be classified as coordinate bonds (and thus identified using an ar- row) to distinguish them from significantly less covalent HBs and other weak interactions.

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The development of novel antimicrobial peptides and various strategies to improve their activity and biocompatibility (2018)

With the advent of antibiotic resistance and crisis, it is crucial to find substitutes to conventional antibiotics. Antimicrobial peptides (AMPs) are considered to be viable alternatives, because they are broad spectrum and bacteria develop little or no resistance towards AMPs. Interestingly, only few AMPs are used as therapeutics, due to problems such as host toxicity, protease cleavage and short half-life. Therefore, there is a need to improve the efficacy of AMPs by the use of D-peptides and/or delivery vehicles. The introduction of the thesis describes the diversity and various mechanisms of action (MOA) of AMPs. The issues and ways to improve the efficacy of AMPs, which forms the foundation of this thesis, are also discussed.Recently, hyperbranched polyglycerol (HPG) has gained attention due to its excellent biocompatibility, multifunctionality and long blood circulation time. The body of the thesis describes a methodology to covalently attach aurein 2.2 and its mutants to HPG and study the influence of the molecular weight on the antimicrobial activity. A peptide array was used to design tryptophan and arginine mutants of aurein 2.2. Mutant peptide 77 had significantly superior antimicrobial and antibiofilm activity compared to aurein 2.2 but was more toxic. We found that HPG can be used as a general scaffold to alleviate the toxicity of the peptides. The conjugates/peptides were tested in an in vivo mice skin infection (abscess) model. Surprisingly, peptide 73 and aurein 2.2 has similar efficacy in vivo indicating both the antimicrobial activity and toxicity, i.e. therapeutic index, are important. The conjugates (HPG-73c) were not active in mice abscess model, whereas 73c and D-73 encapsulated in micelles composed of DSPE-PEG2000 had excellent activity suggesting the release of the peptide from the delivery vehicle is necessary for in vivo activity. Without encapsulation D-73 was too toxic. A bacterial expression system was used to produce isotopically (¹⁵N) labeled aurein 2.2 and its interaction with whole bacterial cells was examined by nuclear magnetic resonance (NMR) and scanning electron microscopy (SEM) confirming the MOA. Finally, the results presented will be discussed in the broad context of designing AMPs for therapeutics and understanding their MOA.

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Protein Interactions of Membrane Protein U24 from Roseolovirus and Implications for Its Function (2016)

This dissertation describes the investigation of the interactions between the tail-anchored membrane protein U24 from Human Herpesvirus type 6A (HHV-6A) and type 7 (HHV-7) and its potential binding partners. The roles that these interactions play in U24s’ function will be presented. It has been suggested that U24 from HHV-6A (U24-6A) may trigger an autoimmune reaction in multiple sclerosis (MS), through its molecular mimicry of myelin basic protein (MBP). Both versions of U24 have been implicated in endocytic recycling via specific binding partners, namely WW domains. The first part of this thesis is a review of the foundations that this thesis is based on, from the description of Roseoloviruses, associated diseases to molecular characterizations. The two main techniques used in the studies will be described as well. Prior to commencing any structural or interaction studies, a protocol is devised to express and purify recombinant U24 from HHV-7 (U24-7), as well as preliminary studies to prepare samples suitable for structure determination by nuclear magnetic resonance (NMR) spectroscopy. U24-6A was shown to be a mimic of MBP and it was suggested that it could be implicated in MS by competing with MBP for its interactions, such as the interactions with Fyn-SH3 domain. The interactions between U24-6A and Fyn-SH3 domain were therefore probed and found to be weak, calling into question this mimicry hypothesis. Because of the weak binding with Fyn-SH3 domain, alternative functions and binding partners were then explored. WW domains were chosen because their binding ligand, the PPxY (PY) motif, is present in U24 and was identified to be essential for U24’s function. In the next part of this thesis, the investigation of the interactions between U24s and WW domains in Nedd4, which is a key component required for endocytosis, are described. U24-7 and phosphorylated U24-6A were found to bind strongly to Nedd4-WW domains, suggesting the negative charge upstream from PY motif in U24 is important for high affinity interactions. Non-canonical Smurf2 WW domains were explored as well. Finally, the results presented in this thesis will be discussed in the context of the function of U24.

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Isolation and characterization of recombinant U24, a membrane protein from human herpesvirus type 6 (2011)

This thesis describes the isolation and characterization of the U24 membrane protein encoded by Human Herpesvirus type-6 (HHV-6), obtained from an E. coli recombinant expression system. HHV-6 infection has been previously associated with the disease multiple sclerosis (MS), and the U24 protein is of interest because it has a seven amino-acid sequence (PRTPPPS) identical to myelin basic protein (MyBP), a candidate auto-antigen in MS. In the first part of this thesis, I describe the methods that were developed to enable milligram quantities of U24 to be isolated and purified from litre cultures of E. coli. Levels of U24 expressed with a maltose binding protein-hexahistidine fusion tag were particularly enhanced by combinations of low temperature, oxidizing conditions, and/or use of minimal media culture. The significance of these results may be considered useful in application to other difficult-to-obtain membrane proteins. Subsequent chapters of this thesis describe testing the recombinant U24 for potential mimicry of MyBP structure and function. Since the polyproline region in MyBP is now being recognized for its potential in cell-signalling roles that relate to myelin sheath development and structural integrity, I hypothesized that U24 may retain some of the same attributes as MyBP on the basis of identical sequence. Results here suggest that U24 can adopt a polyproline type II helix much like MyBP, which is a structural feature important for engaging in protein-protein interactions. Furthermore, the region is also found to represent a PX(T/S)P MAPK phosphorylation motif and PXXP-based Fyn tyrosine kinase SH3 binding domain. These observations are of particular relevance since phosphorylated MyBP is particularly decreased in MS patients, and Fyn is critical to myelin development. Like MyBP, results suggest that U24 can be phosphorylated at the equivalent threonine and is also able to bind to the Fyn-SH3 domain. These results support the possibility that U24 interferes with essential myelin regulation pathways on the basis of its sequence shared with MyBP, thereby contributing to a pathological process. I conclude with a presentation of preliminary NMR structural data for U24, as well as review results from a study of U24 in an animal model system. Future directions are also discussed.

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Investigating the structure-function relationship of cationic antimicrobial peptides and lipopeptides (2010)

Antibiotics have been playing a major role in combating bacterial infections for centuries. Since the discovery of modern antibiotics, numerous derivatives have been designed and developed to treat different bacterial infections. Recently, antibiotic resistance has been continuously and increasingly reported. The lack of antibiotic alternatives makes these resistant bacteria become more difficult to eliminate. Antimicrobial peptides constitute a major part of the innate immune system of an organism. Their high activity and little resistance make them ideal candidates for novel antibiotic development.This dissertation focuses on aurein peptides, a class of amphibian cationic antimicrobial peptides from Litoria aurea, and daptomycin, a lipopeptide. We have examined the structure-function relationship of two aurein peptides, aurein 2.2 and aurein 2.3. They were found to adopt alpha-helices and perturb membrane bilayers via mechanisms similar to toroidal pore or toroidal pore/liposome formation in model membranes. We have also designed and inspected the structure-activity correlation of different aurein 2.2 analogues by residue 13-substitutions and N- and/or C-terminal truncations. We have found that residue 13 and N-terminus are required for antimicrobial activity, whereas an N-terminal truncation gives rise to a peptide analogue with immunomodulatory activity in vitro. The effects of membrane composition and model membrane choice have been further investigated. We have found that the peptide behaviour is dependent on different model membranes. We have examined the importance of solvent accessibility in the mechanism of action for daptomycin and found that daptomycin molecules are indeed solvent-exposed in apo- and Ca2+-form and insert slightly into lipid membranes.Taken together, we have developed a set of references for future design of new antibiotics based on aurein peptides. By using this set of references as a starting point, we hope to gain a better understanding of how antimicrobial peptides function from structural and membrane perspectives and design novel antimicrobial agents to combat increasing antibiotic resistance in the future.

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Structural studies of fusion peptide from tick-borne encephalitis virus and nanocrystalline cellulose films (2009)

This thesis describes structural studies on two different systems, namely the fusion peptide (FP) from the tick-borne encephalitis virus (TBEV) and nanocrystalline cellulose (NCC). The first is a small biopolymer composed of 16 amino acid residues, which binds to the target cell membrane and plays a critical role in membrane fusion. The second is a biopolymer composed of a large number of glucose subunits, which has attracted recent interest with regards to the development of new materials.In this thesis, I have established synthetic access to the model FP fragment from TBEV. To our knowledge, the studies in this thesis are the first investigation on the individual Class II FP. The synthetic peptide can induce membrane fusion at acidic pH. Mutational studies showed that replacement of Leu 107 with Thr strongly impaired fusogenic activity, whereas a Phe mutant still retained a significant degree of activity. These results were consistent with activity found in mutant TBE viruses, indicating that the synthetic TBEV FP obtained here can serve as a model fusion system.Previous literature studies have shown that a disulfide bond stabilizes the peptide fold and the full length fusion protein functions as a trimer. I therefore tested the hypothesis that these are also necessary conditions for the FP. The studies on the modified FPs suggest that the lipid-binding portion in the synthetic peptides adopts a similar conformation. However, the disulfide bond promotes β-strand formation in the peptide. Finally, the formation of an artificial trimer enhances the fusogenic activity. In the second part of this thesis, the chiral nematic structure and crystallinity of the NCC films were investigated. The chiral nematic pitch of the NCC films was affected by all of the parameters listed here, namely the hydrolysis conditions, ionic strength, suspension concentration, drying temperature and magnetic field. The crystallinity of the NCC films was influenced by the drying temperature, ionic strength and magnetic field were small. These results suggest that these sample conditions affect the chiral nematic structure and the crystallinity of the NCC films, a finding which will be important in the development of novel cellulosic materials.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Probing the interactions between U24 from HHV-6B and hNedd4L WW and Fyn-SH3 domains (2022)

Multiple sclerosis (MS) is neurological inflammatory disease in the central nervous system (CNS) that affects every 1 in 400 people in Canada. Although it was first characterized over a century ago, the cause of MS remains elusive, as does consequently a cure. Many factors have been identified as possible triggers, including viruses such as the highly prevalent and latent human herpes virus type 6 (HHV-6) – a member of the Roseolovirus family. There are two variants of this virus, namely HHV-6A and -6B, and the protein U24, encoded in both forms, has been found to be implicated in multiple sclerosis.This thesis describes the study of the binding interaction of U24 from HHV-6B (U24-6B) and two protein binding partners: the WW3* domain from human Neural precursor cell expressed developmentally down-regulated protein 4 like (hNedd4L-WW3*) and the SH3 domain from Fyn tyrosine kinase (Fyn-SH3). These interactions help to further define U24s’ function. Previous studies have suggested that U24 from HHV-6A (U24-6A) and HHV-7 are involved in endocytic recycling of key T-cell receptors, a process mediated by WW domains. Moreover, earlier work has shown that U24-6A may be involved in triggering an autoimmune reaction in MS, through its molecular mimicry of myelin basic protein (MBP), a protein that interacts with Fyn-SH3. None of this previous research investigated whether U24-6B, which is highly homologous to U24-6A, functions in the same way.Hence this thesis aims to fill in this knowledge gap. The interaction between U24-6B and hNedd4L-WW3* was investigated using isothermal titration calorimetry (ITC). The effect of the post-translational modification of phosphorylation at residue Thr6 was also probed. The interaction between U24-6B and Fyn-SH3 was characterized using nuclear magnetic resonance (NMR) spectroscopy. Both these studies shed further insight into the function of U24 from Roseolovirus and its potential role in MS.

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Developing releasable antimicrobial peptide-polyethylene glycol conjugates by targeting infection site-associated host matrix metalloproteinases (2021)

The rapid generation of multidrug-resistant (MDR) bacteria has caused bacterial infections to become a global health concern. Antimicrobial peptides (AMPs), or host defence peptides (HDPs), offer a viable solution to these pathogens due to their broad-spectrum activity and low generation of resistance. In addition, many AMPs possess immunomodulatory properties (e.g., anti-inflammatory activity) that may provide a more robust treatment of infection. However, high toxicity and short biological half-lives have greatly limited the production of clinically available AMP therapeutics. Conjugation of the peptides to delivery vehicles such as polyethylene glycol (PEG) has significantly improved these properties but has also been associated with large reductions in antimicrobial activity, making formulation challenging. In this thesis, an enzymatically releasable PEG-AMP delivery system was developed by incorporating a cleavage sequence susceptible to matrix metalloproteinases (MMPs), enzymes released by the host during the inflammatory response to infection, onto an aurein 2.2-derived AMP. N- vs. C-terminal addition of the sequence found the former to best maintain the activity of the AMP after MMP cleavage, likely due to the maintenance of its amidated C-terminus and higher positive charge. Subsequent conjugation of the cleavable AMP to 2 kDa PEG significantly improved the AMP’s blood biocompatibility in vitro but also eliminated its activity until cleaved by isolated human MMP. This activity was mimicked in an in vivo abscess model of high-density methicillin-resistant Staphylococcus aureus (MRSA) infection, where both free peptide and conjugate displayed strong activity confirmed to be dependent on the accumulation of MMPs at the infection site, as non-cleavable D-isomeric counterparts of the compounds showed no activity. Following this, the system was expanded to larger PEG molecules by incorporating a tetraglycine spacer between carrier and MMP cleavage sequence. This spacer enabled cleavage of the AMP when bound to 5, 10, and 22 kDa PEG, not possible for the initial peptide, allowing for further improvements in biocompatibility compared to the 2 kDa PEG-AMP conjugate. Altogether, the enzyme-releasable delivery system developed here may provide a suitable platform for the development of infection site-targeting AMP therapeutics where both high biocompatibility and activity can be achieved.

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Investigating the protein-protein interactions between TAR DNA-binding protein 43 AND p65 subunit of NF-?B (2017)

TAR DNA-binding Protein 43 (TDP-43), a ubiquitous protein that regulates gene expression, has been found to play a crucial role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), in which the disease is characterized by TDP-43 protein inclusion bodies. Relevant literature suggests that the protein either self-aggregates or interacts with various partners to cause this proteinopathy. One of these binding partners was suggested to be the p65 domain of the nuclear factor kappa-B (NF-κB), a transcription protein complex which plays a crucial role in inflammatory and immune responses. It is upon this hypothetical disease pathogenesis that the study of TDP-43 and NF-κB p65 is rationalized. Hence, the first part of the thesis describes the methods that were developed to obtain pure recombinant TDP-43 from an E.coli expression system. Subsequently, the preparation of NF-κB p65 peptides using solid phase peptide synthesis (SPSS) is described in the thesis. Furthermore, the structural conformation of proteins and peptides was explored using molecular dynamics (MD) simulations to predict how they will behave in vivo and also to allow a comparison to in vitro experimentation.

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Probing the Interaction Between Daptomycin and Model Membranes (2015)

Daptomycin is the first approved antibiotic from the lipopeptide family, with antibacterial activity against a wide variety of Gram-positive bacteria. It is composed of 13 amino acids with a fatty acyl chain attached at the N-terminus. Although it has been approved for clinical usage for over two decades and a number of studies have worked on the mechanism, many details of the mode of action of daptomycin still remain to be understood. This thesis focuses on shedding light on: i) how daptomycin binds to membranes and whether it forms well-defined oligomers; and ii) whether once in the membrane, daptomycin causes leakage. We have used photon correlation spectroscopy (PSC) to determine the condition under which fusion among vesicles occurs in the presence of daptomycin. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPC/POPG) liposome is a better system as higher concentrations of Ca²⁺ is required to trigger membrane fusion. Based on these findings of membrane fusion caused by daptomycin, binding and kinetics experiments were conducted to study the interaction between daptomycin and the lipid membrane. The binding between daptomycin or NBD-labeled daptomycin and POPC/POPG or 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPC/DMPG) liposomes is strong, with micromolar dissociation constants. Förster resonance energy transfer (FRET) experiments were conducted under conditions where fusion is not present. The results show that the oligomerization number n is concentration-dependent. A two-stage equilibrium process is proposed when the daptomycin binds and oligomerizes in the membrane, in which daptomycin accumulates firstly in the membrane to form larger oligomers, facilitating further effects which lead to the cell death, such as the lipid extracting effect on the membrane. In addition, fluorescence experiments were conducted to investigate whether daptomycin causes leakage in POPC/PG and DMPC/PG liposomes, with or without the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Experiments using the ion-selective pore-forming aurein peptide were also done, as a control. For stable liposomes, daptomycin does not cause ion permeabilization of the membrane, suggesting that the membrane depolarization resulting from daptomycin is different from pore-formation by the aurein peptide. The work described in this thesis provides evidence leading to a clearer mechanism of action for daptomycin.

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Probing the Interaction between U24 from HHV-6A and Smurf2 WW Domains (2015)

This thesis describes the study of the interaction of the U24 membrane protein, encoded by Human Herpesvirus type-6A (HHV-6A), with WW domains from Smad ubiquitylation regulatory factor 2 (Smurf2) via its PPxY motif. U24 from HHV-6A is of interest because it acts to block endosomal recycling, as mediated by its PPxY motif interacting with WW domain-containing proteins. We have used a multidisciplinary approach to study the interactions between the Smurf2 WW domains and the PPxY motif-containing region of U24. The GST pull-down experiment demonstrated a difference in affinity between WW domains for the full-length U24 protein, prompting binding studies on interactions between the PPxY motif-containing region of U24 and isolated WW3 domain or WW2 and WW3 domains in tandem (WW23). In Chapter 2, studies were focused on interactions between the third WW domain (WW3) of Smurf2, and the 15-mer U24 peptide containing the PPxY motif. NMR studies demonstrated that the PPxY motif of U24 peptide bound to Smurf2 WW3 domain in a similar way as the interaction between Smurf2 WW3 domain and its cognate ligand Smad7. The dissociation constant was determined to be 123 ± 4 μM at 5°C, reflecting weak binding affinity between WW3 domain and U24 peptide, possibly due to the lack of additional interactions between WW3 domain and the region of U24 peptide beyond the PPxY motif. Circular Dichroism (CD) experiments suggested that isolated WW3 domain was not as stable as other WW domains and binding to U24 peptide enhanced its stability slightly. In Chapter 3, interactions between tandem Smurf2 WW23 and U24 peptide were studied. NMR studies demonstrated that the interaction between Smurf2 WW23 and U24 peptide mainly relied on the U24 peptide binding to its WW3 domain and that the WW2 domain played only a minor role in the interaction. CD experiments were also carried out to detect the stability of Smurf2 WW23 with and without U24 peptide.

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Expression and structural characterization of the p8 major coat protein from B5 filamentous bacteriophage (2011)

This thesis is based on work done on the expression, purification and structuralcharacterization of the major coat protein of B5 bacteriophage. The major coat proteins ofbacteriophages have long been utilized to understand membrane proteins and membrane-associated assembly. It is the special feature of the major coat protein to exist in differentenvironments that holds the key to its involvement in phage assembly. The structure of p8 inthe different environments, especially in the host membrane, has to be fully understoodbefore the mystery of phage assembly can be solved. The major coat protein of B5, p8, hasbeen chosen in this study because B5 infects Gram positive bacteria and the structure of p8 inan appropriate model membrane can better represent its native structure in the hostmembrane.In Chapter 1, I introduce background information on filamentous phage, and thedebate of major coat protein structure. The different structures that already exist for the majorcoat protein in virion, in host membranes, and during phage assembly are discussed.In the next chapter, I present the steps required to obtain pure p8 using a heterologousbacterial expression system. The optimizations and considerations needed to express andpurify p8 are discussed thoroughly. The considerations taken for p8 expression canessentially be applied to other membrane protein expression. In the same chapter, an I32Cmutant of p8 is also designed, expressed and successfully purified. The technique used tointroduce the single substitution mutation to p8 can be applied to other protein mutationexperiments.In the subsequent chapter, p8 structure is studied using circular dichroism (CD),nuclear magnetic resonance (NMR) and site directed labeling with a 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) fluorescence probe. The results from CD show that p8has high alpha helicity when reconstituted into lipid compositions that represent the Grampositive membrane. Preliminary NMR experiments have been performed and conditions toobtain optimal NMR spectra have been explored. BADAN fluorescence labeling experimentshave been trialed and have been shown to successfully indicate the local environment ofresidue 32 to which BADAN is attached. Finally, possible future work is discussed.

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