Many bacteriophages and eukaryotic viruses, which share little sequence similarities, have icosahedral protein capsids containing their genetic materials. Generally, an icosahedral viral capsid is assembly of 12 pentamers and a certain number of hexmers of the major capsid protein, following Caspar and Klug¡¯s quasi-equivalence rule. The arrangement of these pentamers and hexmers is characterized by the triangulation (T) number. Questions arise whether viruses have evolved from a few common ancestors, and how the assembly of the icosahedral capsids has been regulated to achieve a defined capsid size and geometry. I present studies of the capsids of several large icosahedral bacteriophages, which broaden our understanding of the regulation of viral capsid assembly. Bacteriophage SPO1 may share common ancestry with herpesvirus, according to the similarities in their T numbers and in the asymmetric molecules slightly off the local three-fold symmetry positions on the outer surface of both capsids. However, the cryo-EM structure of the SPO1 capsid assembled from the uncleaved major capsid protein show that, unlike the herpesvirus asymmetric molecule, the SPO1 asymmetric protein may not be required for the initial procapsid assembly, suggesting that the two asymmetric molecules may have different origins. Phage P1 is excellent for studying size determination in viral capsid since it produces virions of three sizes. The cryo-EM structures of the three capsids and internal capsid proteins identified suggests a control mechanism for P1 capsids, in which the DarA protein functions as a semi-scaffolding protein to assist the assembly of the P1 big capsid. Jumbo phages have been rarely studied. The structural studies on four jumbo phages showed their T numbers. N3, PAU and 121Q are the first T = 19, 25 and 28 viral capsids found. These results suggest that T-numbers larger than 16 may generally be allowed.

The nicotinic acetylcholine receptor (nAChR) mediates fast signal transduction in peripheral and central nervous systems. It is a pentameric ion channel that belongs to the Cys-loop receptor superfamily. nAChR is one of the plausible targets for general anesthetics. The current concern about nAChR is ambivalent structures of the transmembrane (TM) domain, missing information for the intracellular (IC) domain and elusive mechanisms of the action of general anesthetics. Therefore, this thesis focuses on the following two important aspects: first, the structure and dynamics of the nAChR TM and IC domains; and second, general anesthetic effects on various proteins. Part I. The secondary structure of human nAChR α7 TM domain and the monomeric tertiary structure of the water-soluble mutant of nAChR α1 TM domain from the Torpedo electrical ray were determined by NMR. The structures among WSA, nAChR α7, and a bacterial analogue of the pentameric channels (GLIC) are similar, but different from the cryo-EM structure of the nAChR. The backbone dynamics analyses of human nAChR α7 with and without the IC domain suggest that the presence of IC domains dramatically affected the intrinsic dynamics of the TM domains. Part II. The general anesthetic effects on the structure and dynamics of the proteins including two analogues and a real anesthetic target in non-channel and channel-like oligomers were studied. It revealed that general anesthetics prefer amphipathic environments. Compared to the effect on the structures of proteins, general anesthetics show stronger effect on the dynamics. The effect on the dynamics of proteins can manifest directly or allosterically. Part III. The anesthetic effects on the Na+ flux through the reconstituted channels of nAChR TM domains were investigated by fluorescence microscopy. The anesthetic effects on them are the same as on the full-length nAChR. This indicates that the studies of anesthetic effects on the TM domains of nAChR are functional relevant. In summary, this dissertation clarifies the current structural ambiguities, provides additional invaluable dynamic information for the TM and IC domain and reveals the possible mechanism of action of general anesthetics.

The cytoplasmic dynein motor protein complex transports a number of different important cargos along microtubules (MTs) in a retrograde manner. Cytoplasmic dynein plays an important role in many cellular processes and a number of diseases have been associated with defects in its activity. Despite its importance, there are no small molecules that selectively modulate cytoplasmic dynein activity, nor is its atomic structure elucidated. In an effort to identify compounds that target cytoplasmic dynein, hits from a high information content cell-based nuclear translocation assay were further evaluated biochemically. High throughput assays were developed to screen for glucocorticoid ligand competition, MT perturbation, and the ATPase activities of Hsp 70 and 90, cytoplasmic dynein heavy chain 1, and myosin. Several compounds from screening the Library of Pharmacologically Active Compounds (LOPAC1280) were identified to inhibit cytoplasmic dynein, though they had several unattractive pharmacological properties and were generally non-specific. Additional screening of the Molecular Libraries Screening Center Network >220,000-member library showed a number of compounds that specifically inhibited the ATPase activity of cytoplasmic dynein heavy chain 1 with little or no interaction with other proteins involved in cargo complex formation. A novel approach to screen for MT perturbing agents was also developed using biosensors. Thickness, mass, and density measurements from dual polarization interferometry suggested the growth process of MTs on surfaces. Resonant mirror biosensors were used to distinguish MT stabilizers from destabilizers based on rates of MT assembly on the surfaces. In addition, the structure of the cytoplasmic dynein heavy chain motor domain was characterized by computational and experimental methods. Comparative homology structural modeling was used to predict 15 surface accessible cysteines, which were then correlated experimentally by mass spectrometry. Five cysteines were matched computationally and experimentally to be surface-accessible, suggesting some inadequacy of the proposed model. Finally, attempts to reconstruct a model of cytoplasmic dynein heavy chain 1 by electron microscopy were hindered by the purification of the protein from both a Hi5/baculovirus expression system and bovine brain, although the latter appeared to provide better quality micrographs. Ultimately, structural characterization will assist with the discovery of cytoplasmic dynein heavy chain 1 modulators.

Metabotropic glutamate receptor subtype 6 (mGluR6), a class C G protein coupled receptor (GPCR), plays a key role in visual signal transduction and is also implicated in addiction. Certain mutations in mGluR6 have been reported to cause congenital stationary night blindness. In spite of the importance of mGluR6, knowledge of the molecular basis of its function is lacking. It is imperative to improve the current understanding of its structure-function relationships, so that selective ligands that modulate its activity can be discovered. Furthermore, functional characterization of mGluR6 is also expected to lead to a better understanding of the general principles underlying the activation mechanism of GPCR family. Rhodopsin is the prototypical class A GPCR and serves as a good comparative model to establish general mechanistic patterns of activation of GPCRs. This thesis describes experimental and computational approaches to characterize the structure-function relationship of mGluR6 and its comparison with rhodopsin. Firstly, inducible stable cell lines with high levels of mGluR6 expression were established. Proper trafficking and folding of mGluR6 in these cell lines were verified. To determine mGluR6 function, existing cell-based and novel membrane-based functional assays were optimized and developed, respectively. These efforts led to the establishment of a robust system that expresses properly folded and functional mGluR6 and enabled structure-function studies to be carried out. Several transmembrane cysteine mutants were created and analyzed with the goal to study the role of the transmembrane domain of mGluR6 in activation mechanism. TM6 of mGluR6 like rhodopsin was found to play a key role in its activation supporting the hypothesis that these two GPCRs may share a general mechanism of activation despite the large sequence divergence. Additional support for this hypothesis was obtained from computational sequence analysis which showed that the highly ranking residues involved in long-range interaction in rhodopsin overlap with the allosteric binding pocket of mGluR6. Finally, with the aim to identify selective ligands for mGluR6, an integrated computational-experimental approach was undertaken. Novel allosteric ligands and possibly selective orthosteric ligands for mGluR6 were identified. Further characterization of these ligands may lead to design of selective ligands for mGluR6.

Screening a library of 5,292 hydrazone/hydrazine compounds for inhibition of HIV reverse transcriptase-associated ribonuclease H (RNH) activity identified fluoren-9-yildene hydrazines as highly active inhibitors. The 33 fluoren-9-yildene hydrazines in this library were expanded to 118 compounds, 65 (55%) of which showed validated inhibition of RT RNH activity (IC50 values < 10 uM). These inhibitors were mainly monofunctional for RNH activity, since only 25 (21%) also inhibited RT RNA-dependent DNA polymerase activity. The two most potent RNH inhibitors (RNHIs) were compounds 15 and 25, which inhibited wild type RT-RNH activity with IC50 values of 0.34 ± 0.07 uM and 0.4 ± 0.03 uM, respectively. Similar inhibition was noted with two clinically relevant NNRTI resistant mutants, Y181C and K103N/L100V. Biochemical studies showed that these compounds preferentially inhibited non-directed and DNA 3'-end directed RNH cleavages. These compounds also inhibited the activity of the p15-EC RT RNH domain fragment with IC50 values of 0.43 ± 0.04 uM and 0.032 ± 0.004 uM, respectively. Furthermore, both compounds had antiviral activity against HIV-1 with EC50 values of 10 ± 3 uM and 1.4 ± 0.6 uM for compounds 15 and 25, respectively. Order of addition experiments showed that potent inhibition required pre-incubation of the enzyme with the inhibitor; inhibitory potency substantially decreased if the RNA/DNA substrate was present prior to inhibitor addition. Furthermore, inhibition was competitive with respect to the RNA/DNA substrate, suggesting an active site binding mode. 1H-15N HSQC protein NMR studies with the p15-EC RT RNH domain fragment further suggested that the inhibitor binds to the RNH active site. Molecular docking studies with compound 25 were consistent with an active site binding mode in which the hydrazine functionality hydrogen bonds with essential catalytic metal coordinating residues E52 (RT: 478) and D72 (RT: 498). A sulfonamido-phenyl ring substituent on compound 25 makes edge on-Ï interactions with H127 (RT: 539), another residue essential for RNH catalysis. We therefore propose that the fluoren-9-ylidene hydrazine RNHIs act by preventing access of RNH essential catalytic residues to the RNA/DNA substrate.

The majority of chloroplast proteins are nuclear encoded and transcribed on cytosolic ribosomes, and therefore must be post-translationally imported into the chloroplast. Preproteins are directed to the chloroplast via a cleavable Nterminal extension known as a transit peptide. This transport is mediated by the Toc and Tic complexes (Translocon at the Outer/Inner Chloroplast envelope membrane), functioning in tandem to transport preproteins into chloroplasts relying on the hydrolysis of ATP and GTP. The Toc complex is composed of the β-barrel channel protein Toc75 and the homologous GTPase receptors Toc34 and Toc159. GTP hydrolysis is necessary for the formation of the early import intermediate, in which the transit peptide is inserted into the Toc channel, but the presence of internal ATP is the only energetic requirement for the later stages of translocation to occur, mediated by the stromal motor complex with an Hsp100 isoform hydrolyzing stromal ATP. The purpose of the current study is to characterize the change in stability and/or oligomeric status of the Toc complex with the incubation of nucleotides, analogs, proteins/peptides, etc. by blue native electrophoresis followed by 2d SDS-PAGE. The Toc complex ranges from ~800 kDa to greater than 1320 kDa for the proposed Toc/Tic supercomplex when no proteolytic degradation has occurred. Proteolytic degradation of Toc159 is correlated with the appearance of complexes with a mass ranging from 800 kDa to 440 kDa and below. Proteolytic degradation of Toc159 is more apparent in chloroplasts purified from older Pisum sativum plants. The results of the incubation of chloroplasts with GDP, GTP, and non-hydrolyzable analogs before analysis by 2d electrophoresis followed by western blot hybridization suggest that the loading of the GTPase receptors with nucleotide triphosphate results in the increased association of Toc components in complexes in the size range of 880- 630 kDa.

A neural network (NN) was trained on amino and nucleic acid sequences to test the NN’s ability to predict the correct codon given only an amino acid sequence. Different network configurations were used with varying numbers of input neurons that represented amino acids and a constant representation for the nucleic acid. A multi-layer backpropagation network of one hidden layer with 5 to 9 neurons was used. In the best-trained network, 93% of the overall bases, 85% of the degenerate bases, and 100% of the fixed bases were correctly predicted. The training set was composed of up to 60 human sequences in a window of up to 25 codons at the coding sequence start site. Different input configurations for amino acid representations were designed and evaluated for usage in a large scale NN. This genetic data analysis effort will assist in understanding human gene structure. Benefits include computational tools that could predict more reliably the backtranslation of amino acid sequences useful for Degenerate PCR cloning, and may assist the identification of human gene coding sequences (CDS) from open reading frames in DNA databases.

This study examined the ocular actions of an opioid agonist. Experiments were performed to evaluate the effects of DPDPE ([D-pen2, D-pen5] enkephalin), a delta opioid agonist on: 1) intraocular pressure (IOP) in rabbits; 2) cAMP accumulation in rabbit iris ciliary bodies (ICBs); 3) 3H-norepinephrine (NE) overflow from electrically stimulated sympathetic nerves in ICBs. DPDPE Lowerd IOP in normal rabbits but not in sympathectomized (SX) eyes. Naloxone did not inhibit the effect of DPDPE on IOP in normal rabbits. DPDPE inhibited 3H-NE overflow and suppressed cAMP accumulation in ICBs. The presence of naltrindole, a delta receptor antagonist, did not prevent the suppression of cAMP levels by DPDPE. Pertussis toxin (PTX) did not prevent the inhibition of cAMP levels by DPDPE. The data suggest that the lowering of IOP by DPDPE is mediated at both pre- (neuronal) and postjunctional (ciliary body) sites and may involve an atypical opioid receptor. In addition, the actions of DPDPE in the anterior segment may involve a PTXinsensitive G protein.

The mammalian endoplasmic reticulum (ER) is the largest organelle in the cell, extending from the nuclear envelope throughout the cell periphery. The ER houses a wide variety of vital cell processes within a single membrane bound organelle. In order to accommodate these functions and respond to the demands of the cell, the ER is partitioned into dynamically regulated subdomains, each with its own distinct structure. Despite the likely importance of ER structure for its functions, few proteins have been identified as having a direct role in maintaining the structure of the ER and the consequences of alteration of normal ER structure are not well understood. Here we identify Yip1A, a conserved membrane protein that cycles between the ER and early Golgi, as a likely regulator of ER organization. Yip1A depletion led to restructuring of ER membranes into micrometer-sized, concentrically stacked whorls. These structures are reminiscent of the ER whorls found in certain specialized secretory cell types, where the regulation and functional consequence of ER whorl formation is not understood. We found that membrane stacking and whorl formation after Yip1A depletion coincided with a marked slowing of coat protein (COP) II-mediated protein export from the ER. Furthermore, whorl formation driven by exogenous expression of an ER protein with no role in COPII function also delayed cargo export. Thus, it appears that Yip1A is required to prevent ER whorl formation and that whorl formation can in turn delay protein export from the organelle. Whether this is the function of ER whorls in tissues remains to be seen, however these results make Yip1A a good candidate for playing a role in their regulation. To obtain insight into how Yip1A regulates ER whorl formation and to determine whether the mechanism might be shared with the yeast homologue Yip1p, we carried out a systematic mutational analysis of all residues in the protein. Two discrete sites (E95 and K146) were crucial for the control of ER whorl formation by Yip1A. Notably, the same residues were previously shown to be important for Yip1p-mediated viability in yeast, indicating a shared mechanism. On the other hand, a third site (E89) also essential for yeast viability was dispensable for Yip1A function in regulating whorl formation. Thus Yip1p/Yip1A may possess at least two distinct essential functions only one of which is required for regulation of ER structure. Of note, the sites required for control of ER whorl formation by Yip1A were dispensable for the binding of Yip1p to its established binding partners Yif1p and Ypt1/31p, whereas the site required for Yip1p to bind the same partners was dispensable for ER structuring by Yip1A. Based on these observations, we speculate that the function of Yip1A in regulating whorl formation is mediated by one or more distinct and yet-to-be identified binding partners. Collectively, these findings indicate that a dispersed ER network is important for proper COPII-mediated protein export and that Yip1A has a conserved function between yeast and humans in maintaining proper ER network dispersal through prevention of ER whorl formation. These studies set up an important framework for determining the molecular mechanism of Yip1A as an ER structuring protein

Carbohydrates are important in a large number of cellular, physiological, and pathological processes. Carbohydrates often function as the human host’s first line of defence against pathogen invasion by coating surfaces of epithelial cells and as glycan-rich mucins which line the entrances to the body. Various pathogenic bacteria exploit their hosts by modifying their glycans through the production of carbohydrate-active enzymes. Two kinds of pathogenic bacteria that are notable for their production of carbohydrate-active enzymes are Streptococcus pneumoniae and Clostridium perfringens. Both S. pneumoniae and C. perfringens inhabit glycan-rich niches in the human body, the respiratory and gastrointestinal tracts, respectively. To properly colonize their human hosts both bacteria have developed an extensive repertoire of glycoside hydrolases (GHs) which are enzymes responsible for the breakdown of carbohydrates. These GHs have known or predicted specificities for human glycans, specifically those found in mucins. We chose C. perfringens and S. pneumoniae as model systems to study these enzymes due to their large complements of GHs, many of which are known virulence factors. The objectives are to probe the key features of the GHs from these two different kinds of bacteria that inhabit similar human niches and to study catalysis, modularity and overall enzyme structure. This work uses a multidisciplinary approach and provides molecular level insight into the S. pneumoniae and C. perfringens host-pathogen interaction. / Graduate