During the synthesis of secretory and membrane proteins, polypeptides move through a universally conserved protein-conducting channel, formed by the Sec61/SecY complex that is located in the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. The channel operates in two different modes depending on its binding partners. In co-translational translocation, a pathway found in all organisms, the channel associates with a translating ribosome. In post-translational translocation, the channel cooperates with either the Sec62–Sec63 complex in eukaryotes or the SecA ATPase in bacteria. Despite tremendous progress in our understanding of protein translocation over the past decades, many questions about its mechanism remain to be answered. These include (1) how the channel maintains the membrane barrier for small molecules while transporting large proteins, (2) what is the functional implication of channel oligomerization, and (3) how the channel interacts with binding partners and polypeptide substrates during translocation. To address these questions, we developed a novel in vivo method to generate both co- and post-translation translocation intermediates in intact Escherichia coli cells, such that polypeptide chains are only partially translocated through the channel. Using this method, we first demonstrated that a translocating polypeptide itself blocks small molecules from passing through an open SecY channel. A hydrophobic pore ring surrounding the polypeptide chain is vital for maintaining the membrane barrier during translocation. Next, we examined the importance of SecY oligomerization in protein translocation. Crosslinking experiments showed that SecY molecules interact with each other in native membranes, but that this self-association is greatly decreased upon insertion of polypeptide substrates. We also showed that SecY mutants that cannot form oligomers are still functional in vivo. Collectively, our data indicate that a single copy of SecY is sufficient for protein translocation. Finally, we isolated an intact co-translational translocation intermediate from E. coli cells and analyzed its structure by cryo-electron microscopy. An initial map shows a translating ribosome containing all three tRNAs is bound to one copy of the SecY channel. Analysis of a large dataset is ongoing in order to understand the structural basis of how the channel interacts with the ribosome and translocating nascent chain.

The Ataxia-telangiectasia mutated (ATM) protein is regarded as the major regulator of the cellular response to DNA double Strand Breaks (DSBs). In response to DSBs, ATM dimers dissociates into active monomers in a process promoted by Mre11-Rad50-Nbs1 (MRN) complex. ATM-deficient cells exhibit signs of chronic oxidative stress, suggesting that ATM plays an important role in the regulation of reactive oxygen species (ROS). I show for the first time that ATM can be activated by oxidative stress directly in the form of exposure to H₂O₂. In vitro kinase assays with purified ATM suggest that the activation by H₂O₂ is independent of DSBs and the MRN complex. In 293T cells, H₂O₂ induces ATM autophosphorylation on serine 1981. p53 and Chk2 are also phosphorylated by ATM after H₂O₂ treatment but not histone H2AX and heterochromatin protein Kap1, indicating that ATM activation by H₂O₂ in human cells is independent of DNA damage. I also show that the cysteine residue 2991 is critical for ATM activation by H₂O₂ in vitro. / text

Biometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitious ligands is prevented. In summary, these findings show that the structure and metal site of CusF are unique and are specifically designed to perform the function of CusF as a metallochaperone to the Cus system.

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).

Many protein-protein interactions, especially those involved in eukaryotic signalling, are mediated by PDZ domains through the recognition of hydrophobic C-termini. The availability of experimental PDZ interaction data sets have led to the construction of computational methods to predict PDZ domain-peptide interactions. Such predictors are ideally suited to predict interactions in single organisms or for limited subsets of PDZ domains. As a result, the goal of my thesis has been to build general predictors that can be used to scan the proteomes of multiple organisms for ligands for almost all PDZ domains from select model organisms. A framework consisting of four steps: data collection, feature encoding, predictor training and evaluation was developed and applied for all predictors built in this thesis. The first predictor utilized PDZ domain-peptide sequence information from two interaction data sets obtained from high throughput protein microarray and phage display experiments in mouse and human, respectively. The second predictor used PDZ domain structure and peptide sequence information. I showed that these predictors are complementary to each other, are capable of predicting unseen interactions and can be used for the purposes of proteome scanning in human, worm and fly. As both positive and negative interactions are required for building a successful predictor, a major obstacle I addressed was the generation of artificial negative interactions for training. In particular, I used position weight matrices to generate such negatives for the positive only phage display data and used a semi-supervised learning approach to overcome the problem of over-prediction (i.e. prediction of too many positives). These predictors are available as a community web resource: http://webservice.baderlab.org/domains/POW. Finally, a Bayesian integration method combining information from different biological evidence sources was used to filter the human proteome scanning predictions from both predictors. This resulted in the construction of a comprehensive physiologically relevant high confidence PDZ mediated protein-protein interaction network in human.

Many protein-protein interactions, especially those involved in eukaryotic signalling, are mediated by PDZ domains through the recognition of hydrophobic C-termini. The availability of experimental PDZ interaction data sets have led to the construction of computational methods to predict PDZ domain-peptide interactions. Such predictors are ideally suited to predict interactions in single organisms or for limited subsets of PDZ domains. As a result, the goal of my thesis has been to build general predictors that can be used to scan the proteomes of multiple organisms for ligands for almost all PDZ domains from select model organisms. A framework consisting of four steps: data collection, feature encoding, predictor training and evaluation was developed and applied for all predictors built in this thesis. The first predictor utilized PDZ domain-peptide sequence information from two interaction data sets obtained from high throughput protein microarray and phage display experiments in mouse and human, respectively. The second predictor used PDZ domain structure and peptide sequence information. I showed that these predictors are complementary to each other, are capable of predicting unseen interactions and can be used for the purposes of proteome scanning in human, worm and fly. As both positive and negative interactions are required for building a successful predictor, a major obstacle I addressed was the generation of artificial negative interactions for training. In particular, I used position weight matrices to generate such negatives for the positive only phage display data and used a semi-supervised learning approach to overcome the problem of over-prediction (i.e. prediction of too many positives). These predictors are available as a community web resource: http://webservice.baderlab.org/domains/POW. Finally, a Bayesian integration method combining information from different biological evidence sources was used to filter the human proteome scanning predictions from both predictors. This resulted in the construction of a comprehensive physiologically relevant high confidence PDZ mediated protein-protein interaction network in human.

The baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, thereby enhancing dissemination of progeny virions in nature. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the intracellular processing and trafficking of the baculovirus v-cath expressed cathepsin (V-CATH) is poorly understood and a mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not been determined. The cathepsins of Autographa californica multiple nucleoplyhedrovirus (AcMNPV) and most other group I alphabaculoviruses have well-conserved chymotrypsin cleavage (Y11) and myristoylation sites (G12) suggestive of proteolytic cleavage to generate proV-CATH, and subsequent acylation which could promote membrane anchoring in order to foster cellular retention of the protein. Proteolytic iii N-terminal processing of baculoviral procathepsin was determined by fusing HA epitope-coding tags to the 5’ and/or 3’ ends of v-cath, indicating that the gene is expressed as a pre-proenzyme. However no evidence for myristoylation of proV-CATH was found, suggesting that another mechanism is responsible for retaining proV-CATH in cells. Prior evidence suggested that CHIA is a proV-CATH folding chaperone and that lack of chiA expression causes proV-CATH to become insoluble and unable to mature into V-CATH enzyme. A putative CHIA chaperone activity for assisting in proV-CATH folding implies that proV-CATH and CHIA interact in the ER of infected cells. Fluorescence microscopy demonstrated co-localization of CHIA-GFP and proV-CATH-RFP fusion proteins in the ER. An mRFP-based bimolecular fluorescence complementation (BiFC) assay helped to determine not only that AcMNPV proV-CATH interacts directly with the full-length viral CHIA, but also that it independently binds to the N-terminal chitin-binding domain (CBD) and C-terminal active site domain (ASD) of CHIA, in the ER during virus replication. Moreover, reciprocal Ni/HIS pull-downs of HIS-tagged proteins confirmed the proV-CATH interactions with CHIA, or with the CBD and ASD biochemically. The reciprocal co-purification of proV-CATH with all three polypeptides (CHIA, CBD, ASD) suggests proV-CATH specifically interacts with each of them, and corroborates the BiFC data. Furthermore, CHIA KDEL deletion allowed for premature secretion of not only CHIA but also of proV-CATH, suggesting that the CHIA/proV-CATH interaction in the ER aids cellular retention of proV-CATH. In contrast to prior reports, it was also determined that CHIA is iv dispensable for correct folding of proV-CATH since proV-CATH produced by a chiA-deficient virus was soluble, prematurely secreted from cells and could mature into V-CATH enzyme. Taken together, these data indicate that the viral chitinase plays a major role in ensuring that proV-CATH is neither prematurely secreted nor activated to V-CATH enzyme.

Soy whey protein isolate (SWPI) – Fenugreek gum conjugates were developed and their molecular characteristics and emulsifying properties were investigated. SWPI was extracted from soy whey of tofu processing. SWPI exhibited excellent emulsifying properties comparable to soy protein isolate. However, to improve the emulsifying properties of SWPI for some applications, it was conjugated to fenugreek gum. The extent of conjugation was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared (FTIR) and High performance size exclusion chromatography (HPSEC). The SDS-PAGE of the conjugates showed polydispersed bands at the top of the separating gel in the conjugates suggesting the formation of high molecular weight products. Refractive index spectrum of HPSEC profiles showed a reduction of protein peak of unconjugated mixture and shifted a peak to higher molecular weight of the conjugates. Ultraviolet spectrum of HPSEC showed an increase of protein peak intensity at polysaccharide region. FTIR spectrum showed an amide band I and II were still observed in the conjugates after the unreacted proteins were removed. 1D NMR spectra showed that fenugreek gum was covalently bound to proteins through interaction between the reducing end of mannose residue and lysine. The protein solubility of SWPI – Fenugreek gum conjugates improved as compared to SWPI and SWPI – Fenugreek gum mixture when assessed in the pH range 3 to 8 at 22oC, especially at isoelectric point of protein (pl). A 1:3 and 1:5 ratio of SWPI – Fenugreek gum gave rise to better emulsion stabilization compared to 1:1 ratio. Particle size analysis revealed that conjugation of SWPI – Fenugreek gum at 60oC for 3 days was enough to produce relatively small droplet sizes in oil-in-water emulsions. SWPI – Unhydrolyzed fenugreek gum conjugates exhibited better emulsifying properties compared to SWPI – Partially hydrolyzed fenugreek gum conjugates. The conjugates improved emulsifying properties of SWPI, particularly around the pl of protein. The emulsifying properties were greatly increased by heating the conjugates before emulsification. The conjugates also improved emulsion stability at high salt concentration compared to SWPI. In summary, incorporation of SWPI into fenugreek gum improved emulsifying properties of SWPI near the pl of protein and at high salt concentration. / No / No

Tuberculosis and malaria are among the most deadly infectious diseases in the world. The prevalence in regions without well-established public health causes economical and financial burdens for both society and patients. There is an urgent need to find effective treatments due to the emergence of drug-resistant strains. The aim of the studies reported here was to gain knowledge from the protein structures that can lead to the elimination of these pathogens. In these studies, protein crystallography is the main method used to solve protein structure. Based on the protein structure, we used different methods to characterize the protein function of three lipid-binding proteins (LprG, LprA, and gp232), and to identify potent inhibitors against Plasmodium falciparum enoyl-ACP reductase (PfENR), a drug target protein involved in central lipid metabolism. To characterize the function of two lipid-binding proteins (LprG and LprA), liquid chromatography-mass spectrometry (LC-MS) was used to analyze the ligand extract. In the study of tail fiber protein from mycobacteriophage, we used protein sequence alignment to identify gp232 as a major tail fiber protein, which potentially binds to lipids on the cellular surface of mycobacteria. A pull-down assay and imaging methods (fluorescence microscopy and electron microscopy) were conducted to confirm the function of gp232. In the structural study of PfENR, the structure-activity relationships method was used to find potent inhibitors against PfENR, which would show stronger inhibition than the known inhibitor triclosan. The triclosan-like analogs with modification at the 5-position revealed a new binding site in PfENR that has great potential for improving the potency of inhibition. We found that two inhibitors containing the core structure of piperidine and tetrahydroquinoline reached this new binding site and were 10-fold more potent than triclosan. The structural study of PfENR provides structural insights into the inhibitor-binding site that can lead to the discovery of new drugs. The comprehensive knowledge that we gained from the structural studies of these lipid-binding proteins provide new information that could lead to a greater understanding of pathogen physiology or guide the discovery of effective treatments to eliminate the pathogens.

Accurate 3D protein structure prediction is one of the most challenging problems facing bioinformaticians today. This thesis develops and examines an evaluation module for ranking predicted super-secondary structures – specifically a-helix pairs – as part of a case-based reasoning system. The proposed module is part of the Triptych project, which aims at the accurate prediction of the three-dimensional structure of proteins from contact maps. Triptych is an advanced case-based reasoning system that utilizes a library of existing protein structures and motifs to help predict the structure of a known polypeptide chain of amino acids that represents a target a-helix pair. The proposed module evaluates possible solutions by integrating multiple strategies, learning methods and sources of knowledge in the form of expert advisors. It uses advisors which integrate knowledge from the fields of biology, biochemistry, classical physics, and statistical data analysis obtained from pre-determined structures. Lastly, the proposed evaluation module would allow for the integration of more sources of knowledge, in the form of expert advisors, as well as serve as a framework for evaluating other structural motifs in future. / Thesis (Master, Computing) -- Queen's University, 2007-09-09 19:42:59.094