Elasticity of α-helices and coiled coils have often been described by a linear response to local bending with bending stiffness (Kb) and persistence length (Lp) describing their flexibility. However, we observed that the non-bonded forces along the length of these structures are not screened at physiological conditions and introduce a buckling instability. For α-helical systems of same composition, but different lengths, this is identified by a drop in Kb for longer helices and the length where this drop is triggered is referred to as the critical buckling length. When shorter than their critical buckling length they behave linearly, and Kb calculated using normal mode analysis in this regime is about (3.0−3.4)×10-28 Nm2 for α-helices with varying compositions, and about (1.9 − 2.1) × 10−27 Nm2 for coiled coils with leucine zipper periodicity. Beyond the critical buckling length, normal mode solutions turn imaginary, leading to an eventual disappearance of bending modes. Investigations with one dimensional (1-D) linear chains of beads (a simplistic representation of bio-filaments) show that non-bonded forces have a reciprocal relation with the critical buckling length (no buckling instability existed in the absence of non-bonded forces). Critical buckling length is 115.3 ± 2.9 °A for α-helices and 695.1 ± 44.8 Å for coiled coils with leucine zipper periodicity, which is much smaller than their Lp (~ 800 Å for α-helices and ~ 3000 Å for coiled coils).

The detection of cellular events is central to understanding biomoleculer processes as well as aid in therapeutic intervention strategies. One of the most fascinating biomoleculer events during the life cycle of a cell is proteolytic cleavage of proteins by enzymes known as proteases. Proteases are ubiquitous and participate in essential functions such as fertilization, embryo development, cell cycle regulation, immune response, tissue remodeling and programmed cell death. As proteases are involved in fundamental cellular processes any dysregulation of protease activity is usually associated with a diseased state. Thus methods for detection of protease activity are desirable as it may facilitate the identification of many pathological conditions which are associated with the aberrant expression and activity of proteases.Towards the goal of a general and modular strategy we have utilized split protein reassembly and coiled coils to develop genetically encoded sensors for detection of proteases. We established our first generation protease design utilizing split firefly luciferase and anti-parallel coiled coils and detected Tobacco Etch Virus (TEV) as a model protease. Two further iterations of the coiled-coil design led to the development of second and third generation of protease sensors which showed substantial improvement in the sensor response and was applied towards detection of therapeutically relevant proteases such as caspase-3, prostate specific antigen (PSA), ß-secretase and calpain-1.We applied our methodolgy to develop protease biosensors for the detection of a family of cysteine protease known as caspases. Caspases are involved in programmed cell death and their misregulation is implicated in cancer as well as neurodegenerative disorders. The panel of caspase biosensors was utilized to investigate caspase cleavage specificity as well as caspase activation in mammalian cytosolic extracts and live mammalian cells. Perhaps more importantly, we discovered cross talk between members of the caspase family which perform different biological functions.Finally, we detail our progress towards mimicking a naturally occurring multicomponent complex formed during programmed cell death, known as the apoptosome which leads to the activation of caspases. We have successfully utilized principles of self assembly and multivalency to assemble multi component complexes which exhibit proteolytic activity similar to the natural apoptosome.

Proteins have a wide array of essential functions: from serving as enzymatic catalysts to protecting the immune system as antibodies. Proteins spontaneously self-organize into specific, folded structures determined by their amino acid sequences and the interaction between molecular forces. Since the 3-dimensional structure into which they fold often relates to the specific function of the protein, much effort has been directed towards methods to predict the folded structure from a given sequence, with the hope of being able to understand protein functions from sequence information. The protein folding problem can be summarized as the attempt to understand the relationship between a protein sequence and a protein's geometric shape, or fold. Thus, there are two principal problems: given a sequence, what 3-dimensional form will the protein take (forward problem), and given a particular fold, what sequence or sequences code for that form (the inverse problem). In this work, models that represent folds as continuous structures are explored. Models of the two prevalent motifs in protein folds, α helices and β barrels, are developed using axially deformed tubes and surfaces of revolution. These models are then analyzed and used to develop coordinate models of known and unknown structures.

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Alpha-helical coiled-coils are common protein structural motifs with varied mechanical roles, such as, tropomyosin in muscle contraction or neck-stalks of kinesins and myosins, in motor proteins. Using computer simulations, we characterized elastic properties of coiled-coils both, globally and locally. Normal mode analysis for global elastic properties revealed a buckling instability due to inherently present weak non-bonded forces. We characterized this using a critical buckling length (lc). For coiled-coils, lc was significantly less than their persistence length thereby governing the filament conformation. We also found that mutations to the hydrophobic residues at the knob-into-hole interface affect elasticity of coiled-coils significantly. We built a flexibility map of tropomyosin using a local fluctuation analysis and found regional variations in flexibilities due to such breaks in the knob-into-hole packing. Overall, flexibility varies by more than twofold and increases towards the C-terminal region of the molecule. Actin binding sites in zones and broken core regions due to acidic residues at the hydrophobic face such as, the Asp137 and the Glu218, are found to be the most labile with moduli for splay and broad face bending as 70 nm and 116 nm, respectively. Such variations in flexibility could be relevant to the tropomyosin function, especially for moving across the non-uniform surface of F-actin to regulate myosin binding. Non-claret disjunction (Ncd), is a Kinesin-14 family protein that walks to the microtubule's minus end. Although available structures show its alpha-helical coiled-coil neck in either pre- or post-stroke orientations, little is known about the transition between these two states. Using a combination of molecular dynamics simulations and structural analyses, we find that the neck travel is a guided diffusion involving sequential intermediate contacts with the motor head. The post-stroke is at a higher free-energy minimum than the pre-stroke. The importance of intermediate contacts correlates with the existing motility data including those of mutant Ncds and other members of the kinesin-14 family. While the forward motion has a ~4.5 kBT (kB: Boltzmann constant, T = 300 K) free energy barrier, recovery stroke goes nearly downhill in free energy. The hysteresis in forward and reverse neck motion energetics arises from the mechanical compliance of the protein, and together with guided diffusion, it may be key for the directed motility of Ncd. Although it is known that neighboring Ncds on a microtubule (MT) have an attractive interaction and a group of Ncds act cooperatively, the physical basis of neither this attraction nor the cooperativity is known. From structural analysis of Ncd neighbors on an MT lattice we find that steric hindrances between the coiled-coil neck-stalks of longitudinal neighbors drive synchrony among a group of Ncds on a single protofilament. Across lateral dimers, surface loop L2 of the motor-head (MH) that is not bound to the MT (unbound-MH) in a pre-stroke dimer, is seen to have strong attraction to the nucleotide pocket in the MH that is bound to MT (bound-MH) of its off-axis neighbor. Such an attraction will however impede the motility in both the dimers. We hence propose rules that drive motor binding to an MT site in the presence of immediate neighbors such that motility of the group is not compromised. The unbound-MH, whose role in the walking step of an Ncd was unclear, is thus seen to regulate MT decoration.

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<p> We developed some universal platforms to overexpress the minimal functional entities of ion channels. The modular property of ion channels have been demonstrated from many aspects, such as crystal structures, chimeric channel experiments and discovery of similar modules in distantly related protein families. Thus it should be feasible to express each module independent of other channel modules. The pore-forming module of ion channels has multiple important properties as selectivity, conductivity and drug-binding. If it can be overexpressed, it will provide valuable information about channel selectivity to different ions and structural bases for drug binding as well as important application in drug screening and rational drug design. </p> <p> To test this, we first used the model channel KcsA to identify the minimal requirements for a pore-forming domain to functionally exist independently. Chapter 2 of this thesis explains in detail how the wild type C-terminal cytoplasmic domain of KcsA functions. We found that this domain has dual function as pH-sensor and tetramerization domain, and it is essential for the expression of the pore-forming domain of KcsA. Once we knew the physiological role of the cytoplasmic domain, the scenario was set to answer the question of how to make it better for the application of structural and functional studies. </p> <p> In chapter 3 and chapter 4, we replaced the wild type C-terminal domain with non-native tetramerization domains. We identified the direct correlation between protein expression level and overall thermostability of pore-forming domains. The C-terminal tetramerization domains stabilize channels in a cooperative way and play a critical way in in vivo channel assembly. The selection of the linker between pore-forming domain and tetramerization domain, the splicing motif, and the handedness of C-terminal tetrameric coiled coils all affect channel expression level and stability. </p> <p> We applied our finding in KcsA to a wide range of ion channels in chapter 5, including voltage-gated potassium channels, Ca2+-gated potassium channels, inwardrectifying potassium channels, cyclic nucleotide-gated potassium channels and voltagegated sodium channels. We managed to express similar minimal structural modules from these more structurally complicated channels with the assistance of different cytoplasmic tetramerization domains. Several minimal channels expressed well and showed similar biophysical and functional property as the wild type channels. </p> <p> These studies demonstrate that the pore-forming modules of ion channels can be expressed independently while retaining the proper structure and drug-binding properties as their wild type predecessors when using our universal expression platform. The potential application in structural studies and drug-screening is promising. </p> / Thesis / Doctor of Philosophy (PhD)

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