Rett syndrome (RTT) is an X-linked neurological disorder primarily caused by mutations in the MECP2 gene. The majority of RTT mutations disrupt the interaction of MeCP2 with DNA or TBL1X/TBL1XR1, which forms the scaffold of NCoR/SMRT co-repressor complex. Patients with RTT show no signs of neuronal death, although they have abnormal neuronal morphology, indicating that it is a neurodevelopmental rather than a neurodegenerative disease. It has been shown that reactivation of silenced MeCP2 in mice rescues the RTT phenotype, which implies that the disease is treatable. The RTT mutations in MeCP2 cluster to two regions - the methyl-CpG-binding domain (MBD) and NCoR/SMRT Interaction Domain (NID). While the interaction between MBD and DNA has been biochemically and structurally characterised, there are no structural data about the interaction between MeCP2 NID and TBL1XR1. The aim of this work was to understand how mutations in the NID cause RTT by characterising the interaction between MeCP2 and TBL1XR1. I have solved the structure of MeCP2 NID bound to TBL1XR1 WD40 domain. I show that a small region of the MeCP2 NID makes extensive contacts with TBL1XR1, and that these contacts are mediated primarily by MeCP2 residues known to be mutated in RTT. I also measured the affinities between TBL1XR1 and MeCP2-derived peptides using fluorescence anisotropy and surface plasmon resonance assays. I determined the affinity between MeCP2 NID peptide and TBL1XR1 to be around 10- 20 μM, and show that mutations in either MeCP2 or TBL1XR1 can abolish this interaction. Taken together, these data strongly suggest that the abolition of the interaction between MeCP2 NID and TBL1XR1 WD40 domain is sufficient to cause RTT. This knowledge can help with the rational design of small drug-like molecules that might be able to mediate the interaction between mutated MeCP2 and TBL1XR1, potentially helping to treat the disease.

Helical assemblies of proteins are ubiquitous in nature and they perform vital functions in a wide range of organisms. The recent development of direct electron detectors and other imaging techniques in cryo-electron microscopy (cryo-EM) has opened new possibilities in solving helical structures at atomic resolution. Existing software packages for helical processing often require experience in tuning many ad hoc parameters to achieve optimal reconstruction results. REgularised LIkelihood OptimisatioN (RELION), an open-source single-particle analysis package, reduces the need for user expertise by the formulation of an empirical Bayesian framework, and has yielded some of the highest resolution density maps in recent years. Prior information about the helical assemblies can be conveniently incorporated into the statistical framework of RELION and thereby improves the helical reconstructions. This PhD thesis describes the development of a helical processing computation workflow with reduced user intervention in RELION. Chapter 1 introduces the theoretical basis of cryo-EM data acquisition and single-particle data processing, the concepts of helical symmetry, and a previously described method for iterative real-space reconstruction of helical assemblies, to which the RELION implementation bears resemblance. Chapter 2 discusses multiple adaptations to RELION that are necessary for helical processing. Key elements include the imposition and local refinement of helical symmetry, masks on helical segments and references, expressions of angular and translational prior information, manual and automated segment picking as well as initial model generation for helices. Calculations have been performed on four test data sets showing that the developed methods in RELION yield results that are as good as or better than alternative approaches for the tests performed. Chapter 3 describes the same methodology adapted to helical sub-tomogram averaging in RELION. Chapter 4 introduces the local symmetry option developed for special types of filaments with pseudo-helical symmetry. The concept can be extended to general single-particle analysis as well. Chapter 5 describes four helical structures determined in collaboration with other research groups using helical RELION for data processing. Chapter 6 concludes the thesis with a brief summary and future prospects.

Protein structure prediction is a necessary tool in the field of bioinformatic analysis. It is a non-trivial process that can add a great deal of information to a genome annotation. This dissertation deals with protein structure prediction through the technique of protein fold recognition and outlines several strategies for the improvement of protein threading techniques. In order to improve protein threading performance, this dissertation begins with an outline of sequence/structure alignment energy functions. A technique called Violated Inequality Minimization is used to quickly adapt to the changing energy landscape as new energy functions are added. To continue the improvement of alignment accuracy and fold recognition, new formulations of energy functions are used for the creation of the sequence/structure alignment. These energies include a formulation of a gap penalty which is dependent on sequence characteristics different from the traditional constant penalty. Another proposed energy is dependent on conserved structural patterns found during threading. These structural patterns have been employed to refine the sequence/structure alignment in my research. The section on Linear Programming Algorithm for protein structure alignment deals with the optimization of an alignment using additional residue-pair energy functions. In the original version of the model, all cores had to be aligned to the target sequence. Our research outlines an expansion of the original threading model which allows for a more flexible alignment by allowing core deletions. Aside from improvements in fold recognition and alignment accuracy, there is also a need to ensure that these techniques can scale for the computational demands of genome level structure prediction. A heuristic decision making processes has been designed to automate the classification and preparation of proteins for prediction. A graph analysis has been applied to the integration of different tools involved in the pipeline. Analysis of the data dependency graph allows for automatic parallelization of genome structure prediction. These different contributions help to improve the overall performance of protein threading and help distribute computations across a large set of computers to help make genome scale protein structure prediction practically feasible.

Light is the primary entraining signal for the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Light entering the eye leads to release of glutamate directly onto SCN neurons where it binds to N-methyl D-aspartate (NMDA) receptors initiating a cascade of cellular processes that ultimately modulates clock phase. SCN neurons show a 24-hour rhythm in neuronal activity that peaks in the middle of the day when isolated in a brain slice preparation. Treatments that phase-shift the SCN clock in vivo have been shown similarly to shift this rhythm of neuronal activity in vitro. Here, I have investigated the effects of ethanol on circadian rhythms in SCN brain slices. My experiments have found that acute application of glutamate [1mM] in the early night to mouse SCN brain slices causes a mean phase-delay of 2.7 hours. Co-application of ethanol blocks this phase-delay in a dose dependent manner, with a maximum effect at 20mM. Ethanol could affect the glutamate-initiated pathway through one or more mechanisms including the prevention of glutamate and/or its co-agonists from binding to the NMDA receptor. Experiments, however, show that high levels of glutamate are not able to overcome ethanol’s blocking effects. Similarly, excess concentrations of glycine and D-serine, the two potential co-agonist candidates, do not prevent ethanol’s block. Additional experiments showed that high concentrations of glycine applied in the early subjective night caused a phase-advance of 3.4 hours, indicating the presence of glycine receptors in the SCN that are capable of modulating clock phase. Experiments involving strychnine, a glycine receptor antagonist, revealed that when applied alone in the early subjective night resulted in a phase delay of 2 hours. The mechanism behind strychnine’s phase shifting abilities is unknown. This study has shown that ethanol at a physiologically relevant level has the ability to block glutamate induced phase delays. Though the specific mechanism through which it acts has not been identified, this study suggests ethanol does not interfere with binding of glutamate, nor its co-agonist, to the NMDA receptor. In addition to the ethanol work, my study has shown the phase-shifting effects of glycine and strychnine, two previously unknown phenomena.

Caffeine is found in coffee, tea, soft drinks, many plant products and various drug preparations. It is the most consumed common psychoactive drug around the world. Consumption of caffeine causes several behavioral and physiological responses in humans and other mammals. Caffeine is also known to be an insect repellant and can be used as an insecticide. As observed in mammals, caffeine treatment increases the locomotor activity in insects including Drosophila. However, very little is known about genetic and molecular basis of caffeine sensitivity and action in insects. In the present study, I have used DDT resistant (91-R) and susceptible (91-C and ry506) strains of Drosophila melanogaster to examine whether these strains also differ in caffeine resistance and locomotor activity following caffeine treatment. Results showed that time required for 50% mortality (LT-50) of the 91-R strain were at least 2-fold higher than the LT-50 of the 91-C and ry506 strains. In all strains, caffeine LT-50 was found to be at least 1.5-fold higher in females than in males. I also used chromosome substitution stocks made between the DDT resistant 91-R and DDT susceptible 91-C and ry506 strains. Caffeine-mortality tests on these stocks showed that the major resistance factors against caffeine are linked to the second chromosome and the factors on the X and the third chromosomes play a minor but positive role. Experiments on locomotor activity showed that on caffeine-free media both DDT resistant and susceptible strains were more active during light than dark cycle. While the both DDT susceptible strains showed increased locomotor activity on caffeine media during dark and light cycle, the DDT resistant 91-R strain did not show any change in locomotor activity on medium containing low dose (1.5mM) caffeine. This refractoriness to low dose of caffeine appears to be linked to the second chromosome as deduced by examining the chromosome substitution stocks; strains carrying the second chromosome of 91-R displayed this behavior. On the other hand, locomotor activity of the DDT resistant strain decreased both during light and dark cycle when exposed to higher dose (3mM) of caffeine. This behavior is again found to be linked to the second chromosome because chromosome substitution stocks carrying the second chromosome of the 91-R strain showed decrease in locomotor activity on medium containing 3mM caffeine. The X and 3rd chromosomes also carry factors that modulate the effect of the second chromosome, but in a complex manner.

Chloroplasts are the powerhouses of plants and also perform important storage functions. Chloroplast division is an essential process that involves proteins that are conserved from prokaryotic fission and proteins evolved in eukaryotes. Due to their endosymbiotic origin, the division machineries of chloroplasts and all plastids share some core similarities with the bacterial division apparatus, but during evolution some prokaryotic components of the division machinery were not conserved and some novel components evolved to fulfill new functions. The components of the division apparatus and their interactions are being elucidated, but relatively little is known about the mechanism and dynamics of the first protein families to localize to the division site, FtsZ1 and FtsZ2. This work details a thorough investigation of the biochemical characterization of Arabidopsis thaliana FtsZ proteins and begins to determine the mechanism of FtsZ assembly. To achieve these ends a number of techniques were incorporated including: electron microscopy, protein purification, sedimentation and image processing. Following expression of FtsZ and subsequent purification, experiments aimed at assessing the activity were conducted. These included determining whether the protein was an active GTPase and capable of self-assembly as the bacterial FtsZ homolog displayed these characteristics. The recombinant protein displayed both of these activities and this result allowed for further characterization. The co-assembly critical concentration and assembly efficiency were determined by sedimentation and were 82.75 μg/ml and 33.4 ± 0.9%, respectively. Bacterial FtsZ assemblies have been reported to be in dynamic exchange with a soluble pool of FtsZ and the existence of a similar pool in plants has been discussed in the literature. Chapter III of this work investigates the composition of the soluble pool in Arabidopsis chloroplasts. Gel chromatography revealed that prior to FtsZ assembly initiation the pool consists solely of dimers. Image processing and native PAGE results suggest that at least one assembly intermediate exists between the dimer and mature filamentous assemblies. The most common intermediate observed in assembly reactions is a tetramer. Three-dimensional renderings of the dimer and tetramer are presented in chapter III and suggest that these oligomeric forms may represent consecutive steps in the assembly mechanism of Arabidopsis FtsZ.

This work details the design and generation of a monomer specific p53 rabbit polyclonal antibody. The technique involved careful analysis of published Nuclear Magnetic Resonance(NMR)and X-Ray structures to select an epitope buried in the wildtype tetramer of p53,while exposed in the monomer. The antibody was validated with indirect Enzyme Linked Immunosorbant Assay(ELISA),competition ELISA and western blot. Following these experiments, the antibody, denoted as α-tet,was employed in immunofluorescence (IF) experiments of cancer cell lines DU145 (prostate carcinoma, p53 P223L and V274F) and A549 (lung carcinoma,p53 wildtype)to determine the localization of monomeric p53. Monomeric p53 was confined to the nucleolus of DU-145 cells. Additional staining of the Golgi apparatus in both cell lines was also observed. However, upon investigation of a p53 null cell line, SKOV-3,the Golgi staining was determined to be a result of cross reactivity with another protein. Nevertheless, the presence of nucleolar monomeric p53 in DU-145 cells indicates that monomerization of p53 does occur in this cell line.

This work details the design and generation of a monomer specific p53 rabbit polyclonal antibody. The technique involved careful analysis of published Nuclear Magnetic Resonance(NMR)and X-Ray structures to select an epitope buried in the wildtype tetramer of p53,while exposed in the monomer. The antibody was validated with indirect Enzyme Linked Immunosorbant Assay(ELISA),competition ELISA and western blot. Following these experiments, the antibody, denoted as α-tet,was employed in immunofluorescence (IF) experiments of cancer cell lines DU145 (prostate carcinoma, p53 P223L and V274F) and A549 (lung carcinoma,p53 wildtype)to determine the localization of monomeric p53. Monomeric p53 was confined to the nucleolus of DU-145 cells. Additional staining of the Golgi apparatus in both cell lines was also observed. However, upon investigation of a p53 null cell line, SKOV-3,the Golgi staining was determined to be a result of cross reactivity with another protein. Nevertheless, the presence of nucleolar monomeric p53 in DU-145 cells indicates that monomerization of p53 does occur in this cell line.

The ubiquitination pathway regulates virtually all cellular processes such as cell cycle control and immune surveillance in eukaryotes, and is thus highly regulated through a variety of means. For instance, the Cullin-RING ubiquitin E3 ligases are regulated by neddylation through the action of a newly identified protein Dcn1. In chapter two, I describe an X-ray crystal structure of yeast Dcn1, encompassing an N-terminal ubiquitin association (UBA) domain and a C-terminal domain of unique architecture, which I termed the PONY (POtentiating NeddYlation) domain. I describe the identification of the reciprocal, conserved binding surfaces on both Dcn1 and yeast cullin Cdc53. In collaboration with Dr. Matthias Peter’s group (ETH Zurich), we show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Together, our data identify Dcn1 as the long sought-after Nedd8 E3 ligase for cullin neddylation. As a modulator of immune surveillance and inflammatory responses, the ubiquitin system serves as an attractive target for subversion by pathogens. In chapter three, I present a structural and functional analysis of a newly identified bacterial ubiquitin E3 ligase IpaH, present in various pathogenic and commensal bacteria. I demonstrate that the leucine-rich repeat (LRR) substrate recognition domains of different IpaH enzymes auto-inhibit the enzymatic activity of the adjacent catalytic domain by two distinct but conserved structural mechanisms. Auto-inhibition is required for the biological activity of two IpaH enzymes in a yeast model system. Retro-engineering of auto-inhibition into a constitutively active IpaH enzyme from Yersinia demonstrates that most of the infrastructure required to support auto-inhibition is evolutionarily conserved. In brief, my research provides insights into the mechanism of action of two newly identified protein ligases in the ubiquitination pathway, namely the Nedd8 E3 ligase Dcn1 and bacterial ubiquitin E3 ligase IpaH.

The ubiquitination pathway regulates virtually all cellular processes such as cell cycle control and immune surveillance in eukaryotes, and is thus highly regulated through a variety of means. For instance, the Cullin-RING ubiquitin E3 ligases are regulated by neddylation through the action of a newly identified protein Dcn1. In chapter two, I describe an X-ray crystal structure of yeast Dcn1, encompassing an N-terminal ubiquitin association (UBA) domain and a C-terminal domain of unique architecture, which I termed the PONY (POtentiating NeddYlation) domain. I describe the identification of the reciprocal, conserved binding surfaces on both Dcn1 and yeast cullin Cdc53. In collaboration with Dr. Matthias Peter’s group (ETH Zurich), we show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Together, our data identify Dcn1 as the long sought-after Nedd8 E3 ligase for cullin neddylation. As a modulator of immune surveillance and inflammatory responses, the ubiquitin system serves as an attractive target for subversion by pathogens. In chapter three, I present a structural and functional analysis of a newly identified bacterial ubiquitin E3 ligase IpaH, present in various pathogenic and commensal bacteria. I demonstrate that the leucine-rich repeat (LRR) substrate recognition domains of different IpaH enzymes auto-inhibit the enzymatic activity of the adjacent catalytic domain by two distinct but conserved structural mechanisms. Auto-inhibition is required for the biological activity of two IpaH enzymes in a yeast model system. Retro-engineering of auto-inhibition into a constitutively active IpaH enzyme from Yersinia demonstrates that most of the infrastructure required to support auto-inhibition is evolutionarily conserved. In brief, my research provides insights into the mechanism of action of two newly identified protein ligases in the ubiquitination pathway, namely the Nedd8 E3 ligase Dcn1 and bacterial ubiquitin E3 ligase IpaH.