The key control step in E. coli chemotaxis is regulation of CheA kinase activity by a set of four transmembrane chemoreceptors. The receptor dimers can form trimeric complexes (trimers of dimers), and these trimers can be joined by a bridge thought to consist of a CheW monomer, a CheA dimer, and a second CheW monomer. It has been proposed that trimers of receptor dimers may be joined by CheW-CheA dimer-CheW links to form an extended hexagonal lattice that may be the structural basis of the chemoreceptor patches seen in E. coli. The receptor/CheA/CheW ternary complex is a membrane-spanning allosteric enzyme whose activity is regulated by protein interactions. The study presented in this dissertation investigated intermolecular and intramolecular interactions that affect the chemotactic signal processing. I have examined functional interactions between the serine receptor Tsr and the aspartate receptor Tar using a receptor coupled in vitro phosphorylation assay. The results reveal the emergent properties of mixed receptor populations and emphasize their importance in the integrated signal processing that underlies bacterial chemotaxis. A mutational analysis of the extreme C-terminus (last fifty residues) of Tar is also presented. The results implicate the receptor C-terminus in maintenance of baseline receptor activity and in attractant-induced transmembrane signaling. They also suggest how adaptive methylation might counteract the effects of attractant binding.

The weakly electric fish Sternopygus macrurus emits an electric organ discharge (EOD) composed of a series of pulses. The EOD pulse is mainly shaped by sodium currents. There are two sodium channel α subunits orthologs of the mammalian Nav1.4 expressed in the EO of Sternopygus. Previous studies identified a novel splice variant of the Nav1.4b (Nav1.4bL), in which an extra 51-amino acid occurs in the N terminal end. Nav1.4bL currents inactivate and recover from inactivation significantly faster than Nav1.4bS. The voltage-dependence of steady-state inactivation of smNav1.4bL shifts to hyperpolarized potential. Structural analysis predicts an α-helix in the middle of the extended N terminus. Removal of a proline right after the α-helix significantly slows down current decay but has no effect on channel recovery from inactivation, suggesting inactivation and recovery have independent mechanism. Mutagenesis analysis of the extended N terminus showed that the short helical region, especially the positive charges in the helix, is an important determinant for channel voltage-dependence of steady-state inactivation. However, other residues outside the helical region are required for regulation of fast inactivation and recovery form inactivation. Functional and structural analysis provides evidence for the importance of the C terminus in fish Nav1.4b channel properties. Chimera in which the C terminus of smNav1.4bS was substituted by the human Nav1.4 C terminus, shows an 11 mV positive shift in voltage-dependence of activation and a -16 mV negative shift in inactivation. Deletion of the distal half of smNav1.4bS negatively shifted voltage-dependence of inactivation and significantly accelerated channel recovery from inactivation. In the deletion mutant, the regulation by the N segment is missing. Substitution of the C terminus mutant retains wild type channel inactivation and recovery properties and can be regulated by N segment again. My study provides evidence that the extended N terminus of smNav1.4bL binds the distal part of C terminal tail to modulate channel inactivation properties. This is the first time to show the distal C terminus is involved in channel recovery from inactivation. Studies in the fish sodium channel properties provide useful information to understand function and structure of voltage-gated sodium channels. / text

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Apoptosis is essential for development and the maintenance of cellular homeostasis and is frequently dysregulated in disease states. Proteins of the BCL-2 family are key modulators of this process and are thus ideal therapeutic targets. In response to diverse apoptotic stimuli, the pro-apoptotic member of BCL-2 family, BAX, redistributes from the cytosol to the mitochondria or endoplasmic reticulum and primes cells for death. The structural changes that enable this lethal protein to transition from a cytosolic form to a membrane-bound form remain poorly understood. Elucidating this process is a necessary step in the development of BAX as a novel therapeutic target for the treatment of cancer, as well as autoimmune and neurodegenerative disorders. A three-part study, utilizing computational modeling and biological assays, was used to examine how BAX, and similar proteins, transition to membranes. The first part tested the hypothesis that the C-terminal α9 helix regulates the distribution and activity of BAX by functioning as a "molecular switch" to trigger conformational changes that enable the protein to redistribute from the cytosol to mitochondrial membrane. Computational analysis, tested in biological assays, revealed a new finding: that the α9 helix can dock into a hydrophobic groove of BAX in two opposite directions – in a self-associated, forward orientation and a previously, unknown reverse orientation that enables dimerization and apoptosis. Peptides, made to mimic the α9-helix, were able to induce the mitochondrial translocation of BAX, but not when key residues in the hydrophobic groove were mutated. Such findings indicate that the α9 helix of BAX can function as a "molecular switch" to mediate occupancy of the hydrophobic groove and regulate the membrane-binding activity of BAX. This new discovery contributes to the understanding of how BAX functions during apoptosis and can lead to the design of new therapeutic approaches based on manipulating the occupancy of the hydrophobic groove. The second and third parts of the study used computational modeling to examine how the helical stability of proteins relates to their ability to functionally transition. Analysis of BAX, as a prototypical transitioning protein, revealed that it has a broad variation in the distribution of its helical interaction energy. This observation led to the hypothesis tested, that proteins which undergo 3D structural transitions during execution of their function have broad variations in the distribution of their helical interaction energies. The result of this study, after examination of a large group of all-alpha proteins, was the development of a novel, predictive computational method, based on measuring helical interactions energies, which can be used to identify new proteins that undergo structural transitioning in the execution of their function. When this method was used to examine transitioning in other members the BCL-2 family, a strong agreement with the published experimental findings resulted. Further, it was revealed that the binding of a ligand, such as a small peptide, to a protein can have significant stabilizing or destabilizing influences that impact upon the activation and function of the protein. This computational analysis thus contributes to a better understanding of the function and regulation of the BCL-2 family members and also offers the means by which peptide mimics that modulate protein activity can be designed for testing in therapeutic endeavors.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第25136号 / 理博第5043号 / 京都大学大学院理学研究科化学専攻 / (主査)教授 寺嶋 正秀, 教授 林 重彦, 教授 渡邊 一也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The Rem, Rem2, Rad, and Gem/Kir GTPases, comprise a novel subfamily of the small Ras-related GTP-binding proteins known as the RGK GTPases, and have been shown to function as potent negative regulators of high voltage-activated (HVA) Ca2+ channels upon overexpression. HVA Ca2+ channels modulate Ca2+ influx in response to membrane depolarization to regulate a wide variety of cellular functions and they minimally consist of a pore-forming α1 subunit, an intracellular β subunit, and a transmembrane complex α2/δ subunit. While the mechanisms underlying RGK-mediated Ca2+ channel regulation remain poorly defined, it appears that both membrane localization and the binding of accessory Ca2+ channel β subunits (CaVβ) are required for suppression of Ca2+ channel currents. We identified a direct interaction between Rem and the L-type Cavα1 C-terminus (CCT), but not the CCT from CaV3.2 T-type channels. Deletion mapping studies suggest that the conserved CB-IQ domain is required for Rem:CCT association, a region known to contribute to both Ca2+-dependent channel inactivation and facilitation through interactions of Ca2+-bound calmodulin (CaM) with the proximal CCT. Furthermore, both Rem2 and Rad GTPases display similar patterns of CCT binding, suggesting that CCT represents a common binding partner for all RGK proteins. While previous studies have found that association of the Rem C-terminus with the plasma membrane is required for channel inhibition, it is not required for CaVβ- subunit binding. However, Rem:CCT association is well correlated with the plasma membrane localization of Rem and more importantly, Rem-mediated channel inhibition upon overexpression. Moreover, co-expression of the proximal CB-IQ containing region of CCT (residues 1507-1669) in HIT-T15 cells partially relieves Rem blockade of ionic current. Interestingly, Ca2+/CaM disrupts Rem:CCT association in vitro. Moreover, CaM overexpression partially relieves Rem-mediated L-type Ca2+ channel inhibition and Rem overexpression alters the kinetics of calcium-dependent inactivation. Together, these data suggest that the association of Rem with the CCT represents a crucial molecular determinant for Rem-mediated L-type Ca2+ channel regulation and provides new insights into this novel channel regulatory process. These studies also suggest that instead of acting as complete Ca2+ channel blockers, RGK proteins may function as endogenous regulators for the channel inactivation machinery.

<p>A number of active site mutants of human Alpha class glutathione transferase A1-1 (hGST A1-1) were made and characterized to determine the structural determinants for alkenal activity. The choice of mutations was based on primary structure alignments of hGST A1-1 and the Alpha class enzyme with the highest alkenal activity, hGST A4-4, from three different species and crystal structure comparisons between the human enzymes. The result was an enzyme with a 3000-fold change in substrate specificity for nonenal over 1-chloro-2,4-dinitrobenzene (CDNB).</p><p>The C-terminus of the Alpha class enzymes is an α-helix that folds over the active site upon substrate binding. The rate-determining step is product release, which is influenced by the movements of the C-terminus, thereby opening the active site. Phenylalanine 220, near the end of the C-terminus, forms an aromatic cluster with tyrosine 9 and phenylalanine 10, positioning the β-carbon of the cysteinyl moiety of glutathione. The effects of phenylalanine 220 mutations on the mobility of the C-terminus were studied by the viscosity dependence of k_cat and k_cat/K_m with glutathione and CDNB as the varied substrates. </p><p>The compatibility of slightly different subunit interfaces within the Alpha class has been studied by heterodimerization between monomers from hGST A1-1 and hGST A4-4. The heterodimer was temperature sensitive, and rehybridized into homodimers at 40 ˚C. The heterodimers did not show strictly additive activities with alkenals and CDNB. This result combined with further studies indicates that there are factors at the subunit interface influencing the catalytic properties of hGST A1-1.</p>

A number of active site mutants of human Alpha class glutathione transferase A1-1 (hGST A1-1) were made and characterized to determine the structural determinants for alkenal activity. The choice of mutations was based on primary structure alignments of hGST A1-1 and the Alpha class enzyme with the highest alkenal activity, hGST A4-4, from three different species and crystal structure comparisons between the human enzymes. The result was an enzyme with a 3000-fold change in substrate specificity for nonenal over 1-chloro-2,4-dinitrobenzene (CDNB). The C-terminus of the Alpha class enzymes is an α-helix that folds over the active site upon substrate binding. The rate-determining step is product release, which is influenced by the movements of the C-terminus, thereby opening the active site. Phenylalanine 220, near the end of the C-terminus, forms an aromatic cluster with tyrosine 9 and phenylalanine 10, positioning the β-carbon of the cysteinyl moiety of glutathione. The effects of phenylalanine 220 mutations on the mobility of the C-terminus were studied by the viscosity dependence of kcat and kcat/Km with glutathione and CDNB as the varied substrates. The compatibility of slightly different subunit interfaces within the Alpha class has been studied by heterodimerization between monomers from hGST A1-1 and hGST A4-4. The heterodimer was temperature sensitive, and rehybridized into homodimers at 40 ˚C. The heterodimers did not show strictly additive activities with alkenals and CDNB. This result combined with further studies indicates that there are factors at the subunit interface influencing the catalytic properties of hGST A1-1.

This thesis performs de novo predictions for functionally significant sequence motifs in the Arabidopsis genome under two separate contexts. Each study applies the use of genomic positional information, statistical over-representation and several biologically contextual filters to maximize the visibility of biological signal in prediction results. Numerous literature supported motifs are prevalent in the results of both studies and a number of novel motif patterns possess a strong potential for in planta significance. The first study examines the statistical over-representation of C-terminal tripeptides as a means for identifying eukaryotic conserved protein targetting signatures. Comparative genomics is applied to the analysis of tripeptide frequencies in the C-terminus of 7 eukaryotic proteomes. While biological signal is maximized through the filtering of both simple sequences and homologous sequences present across protein families. The second study introduces a methodology for the effective prediction of transcription factor binding sites in Arabidopsis. A collection of motif prediction algorithms and a novel enumerative strategy are applied to the prediction of cis-acting regulatory elements within the promoters of genes found coexpressed within distinct tissues and under specific abiotic stress treatments. Overall, the analysis identifies 4 known motifs in expected contexts, 5 known motifs in novel contexts and 7 novel motifs with a high potential for biological function.

This thesis performs de novo predictions for functionally significant sequence motifs in the Arabidopsis genome under two separate contexts. Each study applies the use of genomic positional information, statistical over-representation and several biologically contextual filters to maximize the visibility of biological signal in prediction results. Numerous literature supported motifs are prevalent in the results of both studies and a number of novel motif patterns possess a strong potential for in planta significance. The first study examines the statistical over-representation of C-terminal tripeptides as a means for identifying eukaryotic conserved protein targetting signatures. Comparative genomics is applied to the analysis of tripeptide frequencies in the C-terminus of 7 eukaryotic proteomes. While biological signal is maximized through the filtering of both simple sequences and homologous sequences present across protein families. The second study introduces a methodology for the effective prediction of transcription factor binding sites in Arabidopsis. A collection of motif prediction algorithms and a novel enumerative strategy are applied to the prediction of cis-acting regulatory elements within the promoters of genes found coexpressed within distinct tissues and under specific abiotic stress treatments. Overall, the analysis identifies 4 known motifs in expected contexts, 5 known motifs in novel contexts and 7 novel motifs with a high potential for biological function.