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Now that the functioning of microtubules and the actin cytoskeleton has been worked out in enormous detail, the next important task is defining the structure of intermediate filaments that are far behind the other two major skeletal networks due to their inherent resistance to most structural techniques. The evolution of novel structural approaches for flexible proteins is making this possible now. In my thesis I will aim to elucidate the structure and assembly principles of lamin A nuclear intermediate filament protein. To study lamin A, I principally employed chemical cross-linking that allows the capturing of full-length protein structures in solution. I combined this with mass spectrometry approaches to identify cross-linked residues at the various stages of lamin A assembly that were additionally tracked with SILAC labelling and rotary metal shadowing TEM. Unlike previous cross-linking studies on intermediate filaments I use a zero-length self-excluding cross-linking agent EDC that is better tailored for investigation of the polar interactions between multiple unstructured or otherwise flexible charged sequences of lamins. Using this composite approach I interrogated lamin A dimeric and tetrameric assemblies. I elucidated hinge-like properties of the L12 and found indications that L1 and the region containing coil 2A and L2 and the beginning of coil 2B possess properties of linker-like flexibility and of predicted linear α-helical bundle and could act as molecular springs or compression buffers for the nuclear intermediate filaments. Further I confirm the role of the N-terminal unstructured region in lamin A assembly and for the first time show similar role for the C-terminal unstructured region flanking the rod domain of lamin A. Collected data strongly supports the model where both positively charged unstructured regions participate in extensive interaction with acidic rod termini and act as molecular bridges between these in the head-to-tail interface, confirming the uniformity of this principle between cytoplasmic and nuclear intermediate filaments. Formation of these bridges requires conformational change likely happening due to proline residues in the mitotic phosphorylation sites. Finally I suggest a mechanism of regulation of the order of assembly unique to the nuclear intermediate filament where C-terminal unstructured region blocks lateral interactions until it is tethered to the head-to-tail interface. Collected data on the dynamic behaviour of the C-terminal unstructured region and its ability to tether lamin A Ig domain may have far reaching implications for filament assembly and regulation of binding of hundreds of lamin A partner proteins presenting an important step in our understanding of relationship between lamin A structure and function and how altering the former could lead to disease.

The M protein is the major surface-associated virulence factor of Group A Streptococcus (GAS). M protein is predicted to be a parallel α-helical coiled coil, as identified by a heptad repeat pattern with small, hydrophobic residues at the first and fourth positions. To date, over 200 M variants have been identified, however a common trend among M proteins is that they contain regions characterized as non-ideal coiled coils, with both destabilizing residues in the putative dimer interface and skips and insertions in the heptad pattern. Crystallographic studies on M1 revealed two distinct registers at the fibrinogen (Fg) binding sites: a Fg non-binding register 1 and a Fg-binding register 2. These conformations are related by a rotation of one helical face, resulting in the exposure of adjacent faces of the coiled coil. It was previously found that mutations stabilizing register 1 understandably reduced interaction with Fg. However, we now report that mutations stabilizing register 2 also reduce Fg binding. These results suggest that dynamic motion in M1 is required for association with Fg, in a “lure-and-catch” model. This requirement for binding offers a novel explanation for the presence of non-ideal coiled coil structure in M1, a feature that is evolutionarily conserved among M proteins in general.The M1 protein contains three fibrinogen binding sites, one in the A region and two in the B repeats. Both binding sites in the B repeats are essential to establish a supramolecular complex capable of activating neutrophils. However the function of the binding site in the A region is still unknown. In addition to contributing to immune activation, the M1-fibrinogen interaction contributes to immune evasion. Surface recruitment of fibrinogen has been shown to protect against complement deposition and subsequent phagocytosis. We investigated the role of each fibrinogen binding site in conferring protection from phagocytosis.

This dissertation describes the design of synthetic photoactive metalloproteins and their metal binding properties. Four peptides are prepared to study the interplay between peptide backbone and metal ion clusters. The four peptides synthesized are:K IEALEGK CEACEGK IEALEGK-GGY mini-C9C12-GGYQ IAALEQK IAALEQK CAACEQK IAALEQK GGY AQC16C19-GGYQ IAALEQK IAAVEQK CAACEQK IAALEQK GGY AQVC16C19-GGYQ IAAVEQK CAACEQK IAALEQK GGY miniAQVC16C19-GGYStarting from a previously designed peptide C16C19-GGY, a smaller peptide, mini-C9C12-GGY was designed. Mini-C9C12-GGY has the same peptide sequence with C16C19-GGY except that it has less amino acids than C16C19-GGY at N-terminus. It was shown that mini-C9C12-GGY also can bind to Cu(I) to form a tetrameric metalloprotein Cu(I)/mini-C9C12-GGY which displays an intense room-temperature luminescence around 600 nm. It was also shown that the Cu(I) coordination in Cu(I)/mini-C9C12-GGY might change at high loading of the Cu(I). The emission of the Cu(I)/mini-C9C12-GGY follows biexpotnetial decay and can be quenched by a series of [Ru(NH3)5X] (X= chloro, amine, lutidine, pyridine, nicotinamide, and3,5-dimethyl pyridine dicarboxylate). The quenching mechanism is assigned to a photoinduced electron-transfer event by transient spectroscopy. The bimolecular electron-transfer of Cu(I)/mini-C9C12-GGY occurs in the diffusion limit region. Such results support our hypothesis that the hydrophobic condition provided by the coiled coil structure in Cu(I)/C16C19-GGY results in the prohibition of close approach between the donor and acceptor. The effect of the stability of the apopeptide folding on the oligomerization states of the metalloprotein was demonstrated by a series of peptides derived from a dimeric peptide motif: Q(IAALEQK)nGGY (n>3). The peptides are AQC16C19-GGY, AQVC16C19GGY, and miniAQVC9C12-GGY. AQC16C19-GGY and AQVC16C19GGY exists as coiled coil at their apo-states. MiniAQVC9C12-GGY exists as a random coil due to the short peptide sequence compared to AQC16C19-GGY and AQVC16C19GGY. Although the peptides have different free energies of folding at their apo-states, all the resulting metalloproteins after the addition of Cu(I) exist as trimers as determined by ultracentrifugation analysis and/or high performance size exclusion chromatography. These results indicate that metal ions are still the dominating factor in determining the oligomerization structures of metalloproteins in the researched system.

Potassium (K+) channels are membrane-embedded proteins that selectively pass K+ ions in or out of cells in response to a variety of signals, such as membrane potential changes or binding of ligands. In an excitable cell, such as a neuron or cardiac muscle cell, delayed rectifier voltage-gated K+ channels respond to changes in membrane potential to restore the cell membrane to its resting state after an action potential.In vertebrates, voltage-gated K+ (Kv) channels are tetramers of similar or identical subunits arranged around a central conducting pore. While these channels are primarily gated by membrane potential, their biophysical properties are set by the type of subunits in each tetramer and by interactions with other effector molecules, such as membrane phospholipids, calcium-binding proteins, kinases, and scaffolding proteins. In some cases, discrete intracellular domains control the specific assembly of pore-forming and accessory proteins. However, the molecular mechanisms that direct specific assembly of this wide range of components into a functional K channel complex are incompletely understood.Chapter 2 of this thesis establishes the atomic-resolution structure of one such assembly domain from a Kv7 family channel (Kv7.4). This study suggests the structural basis for specific assembly properties and binding of scaffolding proteins by other members of the Kv7 channel family. Additional studies in Chapter 3 explore implications of the Kv7.4 assembly domain structure for oligomerization in other subtypes. The biochemical and functional effects of Kv7 mutations designed to disrupt or enhance assembly domain oligomerization further support the critical role for this domain in specific assembly of these channels.

Human T-cell lymphotropic virus type I (HTLV-1) is a C type retrovirus that is the causative agent of an aggressive T-cell malignancy, adult T-cell leukemia/lymphoma (ATLL). The virus is also implicated in a number of inflammatory disorders, the most prominent among them being HTLV-1 associated myelopathy or tropical spastic paraparesis (HAM/TSP). HTLV-1, like many viruses that cause chronic infection, has adapted to persist in the face of an active immune response in infected individuals. The viral transactivator Tax is the primary target of the cellular immune response and humoral responses are mainly directed against the envelope protein. Vaccination against HTLV-1 is a feasible option as there is very little genetic and antigenic variability. Vaccination regimes against chronic viruses must be aimed at augmenting the immune response to a level that is sufficient to clear the virus. This requires that the vaccine delivers a potent stimulus to the immune system that closely resembles natural infection to activate both the humoral arm and the cellular arm. It is also clear that multicomponent vaccines may be more beneficial in terms of increasing the breadth of the immune response as well as being applicable in an outbred population. As a step in this direction, this dissertation work first describes the identification and evaluation of human T-cell and B-cell epitope based vaccines that are capable of inducing robust immune responses. A novel strategy was designed for delivery of multiple Tax protein derived CTL epitopes into the same antigen presenting cell for the simultaneous priming of anigen specific T-cells. This design allowed antigen processing by cellular proteasomes to efficiently liberate the individual minimal epitopes endogenously for presentation via MHC Class I. This type of construct was effective in vivo in HLA-A*0201 transgenic mice in inducing cellular immune responses against all individual epitopes. A statistically significant reduction in viral replication was observed in mice that were immunized with a multiepitope Tax CTL construct and challenged with recombinant Tax vaccinia virus, by the induction of antiviral IFN-g and cytolysis of infected target cells. This reduction was determined to be the result of immune responses specifically targeting the Tax protein and was dependent on the presence of CD8+ T-cells. Studies were also undertaken to engineer peptides that have a high propensity to fold into native protein like structure for the induction of antibodies that have a high affinity for the complex transient secondary structure adopted by the native protein. A single matrix multicomponent template strategy was applied to design a peptide from the central region of the TM (residues 347-374) that forms a parallel trimeric hairpin structure. Mutational studies have implicated this region to be critical for the fusion process after receptor binding. The template design served to constrain the individual peptide strands at one end and bring them in close proximity to promote hydrophobic interactions to initiate a coiled coil formation. Key mutations in the native peptide sequence were also made to increase hydrophobic interactions. We observed that the mutations combined with the template design resulted in a peptide that had a high helical content in aqueous medium and antibodies raised against the peptide recognized native viral protein with higher affinity compared with the wild type template peptide antibodies. These results suggested the presence of native sequence like conformation. Furthermore, a chimeric peptide incorporating a “promiscuous” T-helper epitope derived from the region of chain reversal in the TM subunit (residues 392-415 of gp21) implicated in the fusion process was tested for its ability to induce neutralizing antibodies. This peptide induced high titered antibodies that were capable of inhibiting HTLV-1 infected cell induced syncytia formation. These data, taken together may have implications in the development of multivalent vaccines against HTLV-1.