Flavoenzymes catalyze a wide variety of biochemical reactions and are commonly observed as electron transport proteins. The redox reactive portion of the enzymes is the isoalloxazine ring system of the flavin cofactor. It is known that the protein environment modulates the redox potential of the flavin, for example, "tuning" its redox potential to favor either a one-electron transfer (electron transfer proteins) or a two-electron transfer (oxidation reactions). This thesis presents an in depth structural study of the flavoenzyme, cholesterol oxidase (EC 1.1.3.6) from Streptomyces sp. SA-COO (SCOA) a multifunctional enzyme that oxidizes and isomerizes 3-beta-hydroxysteroids. This work was pursued in order to further our understanding of the mechanisms through which the protein interacts with the isoalloxazine system and modulates reactivity. Previous kinetic experiments have identified an active site asparagine (N485) and a histidine residue (H447) both of which are critical to the oxidative activity of the enzyme. On an atomic scale the role of the asparagine residue was unknown. Using mutagensis and crystallographic techniques we have characterized this novel N-H ··· pi protein-flavin interaction. SCOA crystals diffract to sub-atomic resolution providing us with a unique view of the protein bound isoalloxazine system. These atomic resolution maps have revealed unexpected structural features that were not previously apparent in the 1.5 A resolution of SCOA. For example, a second narrow pathway leading directly to the isoalloxazine system was discovered, which has provided a more complete mechanistic understanding of the reactions catalyzed by SCOA. Five atomic resolution structures of SCOA at varying pH values are reported. Differences among these structures provide insight into the affect of pH on protein structure and have revealed structural differences resulting from an inadvertent reduction of the cofactor. For example, these stru

Proteins are known to be post-translationally modified. This thesis will discuss arginine methylation, one of the many post-translational modifications that occur within the cell. The enzymes that catalyze this post-translational modification are called arginine methyltransferases. The three main types of methylated arginines include monomethylated arginine (MMA), asymmetric dimethylated arginine (aDMA) and symmetric dimethylated arginines (sDMA). Type I arginine methyltransferases catalyze the formation of MMA and aDMA; Type II enzyme catalyze the formation of MMA and sDMA. Protein arginine methylation has been implicated in the regulation of many different cellular processes, including transcription, cellular localization, protein-protein interaction and signal transduction. / The purpose of this work was to further characterize arginine methylation by identifying new members of the arginine methyltransferase enzyme family in Drosophila melanogaster and to study the effects of protein arginine methylation on novel substrates. I identified and characterized nine homologues of arginine methyltransferases in Drosophila that were named DART1 to DART9, for drosophila arginine methyltransferases 1-9. All nine enzymes are expressed at various developmental stages. I discovered that a substrate of mammalian enzyme protein arginine methyltransferase 1 (PRMT1) can also be methylated by PRMT5. I also identified HIV-1 Tat protein as the first substrate of the novel enzyme PRMT6.

The tertiary interaction of poly(A)-binding protein (PABP), with eukaryotic translation initiation factor 4G (eIF4G) and 3'poly(A) tail of mRNA acts to stimulate translation initiation. Subsequently, the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3) (via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 and Paip1 is regulated by the presence of amino acids through an mTORC1 dependent signaling pathway. This interaction is inhibited by addition of mTORC1 signaling pathway inhibitors; such as rapamycin and wortmannin. Paip1 binds to eIF3g and this subunit can be phosphorylated on either Thr41 or Ser42. However, we find that phosphorylation on these sites is not stimulated by amino acids, nor does it act to enhance eIF3-Paip1 interaction. On the other hand, we show that S6 ribosomal protein kinase (S6K) positively regulates the interaction of eIF3 and Paip1 and we propose that S6K acts as a putative kinase for eIF3. The studies of the regulation of eIF3-Paip1 interaction will lead to better understanding the translation process. / L'interaction tertiaire entre la protéine PABP (poly(A)-binding protein), le facteur d'initiation de la traduction eucaryote 4G (eIF4G), et la queue poly(A) des ARN messagers, stimule l'initiation de la traduction. De plus, l'interaction entre la protéine qui interagit avec PABP, Paip1 (PABP-interacting protein 1), et le facteur d'initiation de la traduction eucaryote 3 (eIF3) (par sa sous-unité g), induit la traduction de façon plus élevée. Dans cette thèse, nous démontrons que l'interaction entre l'eIF3 et Paip1 est régulée par la présence d'acides aminées, et que cette interaction est dépendante de la voie signalétique contrôlée par mTORC1. En effet, les inhibiteurs de cette voie signalétique comme la rapamycin et la wortmannin bloquent l'interaction. Comme Paip1 s'attache directement à la sous-unité g de l'eIF3 et que cette dernière peut être phosphorylée sur la thréonine 41 ou la sérine 42, nous avons étudié le rôle la phosphorylation de eIF3g sur l'interaction entre les deux protéines. Nous démontrons que cette phosphorylation n'est pas stimulée par l'addition d'acides aminées et qu'elle n'induit pas une plus grande interaction entre eIF3 et Paip1. Par contre, nous observons que la kinase S6K (S6 ribosomal protein kinase), régule de façon positive l'interaction entre eIF3 et Paip1, et nous suggérons que cette kinase agit sur eIF3. Cette étude sur les rôles et actions de eIF3 et Paip1 aide à de plus grandes connaissances sur la régulation de l'initiation de la traduction eucaryote.

Clathrin-coated vesicles (CCVs) are responsible for the transport of proteins between various compartments of the secretory and endocytic systems. Clathrinforms a scaffold around these vesicles and is linked to membranes by clathrin adaptors. The adaptor complexes, AP-2 and AP-1, are key hubs in CCV formation at the plasma membrane and trans-Golgi network (TGN), respectively. They recruit cargo, clathrin and a multitude of accessory proteins. The adaptin, or ear, domains play a critical role in recruiting accessory proteins through binding of specific peptide motifs, such as the FXDXF motif. NECAPs are a family of accessory proteins that enrich in CCVs and associate with AP-2 and AP-1. NECAP1 expression levels are highest in the neuronal cells while NECAP2 is ubiquitously expressed. Structural studies of NECAP1 reveal the fold to be part of the pleckstrin homology (PH) domain superfamily and it mimics AP-2 α-ear binding of FXDXF motifs. Therefore the globular region of NECAP is termed the PHear (PH fold with ear-like function) domain. NECAP1 consists of the PHear domain between amino acids 1-133, and a conserved region that is intrinsically unfolded between 134-178, with the entire unfolded region between amino acids 134-275. This thesis has found using nuclear magnetic resonance (NMR) that the NECAP1 construct 1-178 has a two-fold weaker binding affinity to an FXDXF peptide in comparison to the construct 1-133, with binding constants (KD) of 620 micromolar and 330 micromolar respectively. The decrease in binding affinity is attributed to an interaction between the unfolded region and the PHear domain, with a KD between 4000 and 1000 micromolar. / Les vésicules enduites de clathrine (CCVs pour Clathrin-coated vesicles) sont responsables du transport de protéines entre divers compartiments des systèmes sécréteurs et endocytiques. Les protéines clathrine forment un échafaudage autour de ces vésicules ces dernières sont liées aux membranes par des adaptateurs de clathrine. Les complexes d'adaptateurs, AP-2 et AP-1, sont les points de jonction principaux dans la formation des vésicules enduites de clathrine à la membrane de plasma et au réseau transport-Golgi (TGN), respectivement. Ils recrutent la cargaison, la clathrine et une multitude de protéines accessoires. Les domaines adaptin (aussi appelés domaines oreilles) jouent un rôle critique dans le recrutement des protéines accessoires en se liant à des motifs peptides spécifiques, tels que le motif FXDXF. Les NECAPs sont une famille de protéines accessoires qui se concentrent dans les CCVs et qui s'associent à AP-2 et à AP-1. Les niveaux d'expression NECAP1 sont les plus élevés dans les cellules neuronales tandis que NECAP2 est présente unanimement. Des études structurales de NECAP1 indiquent le pli de la protéine fait d'elle un membre de la superfamille des domaines homologues de pleckstrin (pH). Ces dernières ont comme fonction d'imiter le point d'attache du domaine « α-ear » de AP-2 aux motifs FXDXF. Par conséquent, la région globulaire de NECAP se nomme le domaine PHear (PH fold with ear like function, ou pli PH avec fonction similaire au domaine « ear »).Le domaine PHear de NECAP1 est délimité par les acides aminés 1-133. De plus, la proteine comprend une région conservée qui est intrinsèquement non structurée entre les acides aminés 134 et 178; la totalité de cette région non structuré est comprise entière entre les acides aminés 134 et 275. Cette thèse démontre, en utilisant la résonance magnétique nucléaire (RMN), qu'un segment de NECAP1 composé des acides aminés 1 à 178 a une affinité envers un peptide FXDXF deux fois plus faible que celle d'un segment composé des acides aminés 1 à 133. Les constantes KD étaient de 620 micromolaire et de 330 micromolaire respectivement. La diminution de l'affinité de liaison est attribuée à une interaction entre la région non structurée et le domaine de PHear, avec une constante KD entre 4000 et 1000 micromolaire.

The human adenoviral protein E4orf4 has been shown to induce p53-independent cell death in cancer cells. Interaction with the Balpha subunit of protein phosphatase 2A (PP2A) is essential for E4orf4-mediated cell death. PP2A is a trimeric serine/threonine phosphatase, consisting of a structural A subunit, a catalytic C subunit and a regulatory B subunit. Four families of regulatory subunits have been identified to date: B, B', B" and B"'. The B-family subunits, of which there are 4 isoforms, contain 7 WD-repeats and are predicted to fold into a beta-propeller structure with 7 blades. Recent studies have shown that E4orf4 induces death by inhibiting Balpha-specific phosphatase activity of PP2A against certain substrates. Thus, the goal of this project was to study the interaction between E4orf4 and the Balpha subunit of PP2A. To map the PP2A Balpha E4orf4-binding site, point mutants were constructed for Balpha, converting conserved residues amongst E4orf4-binding proteins to alanines. The results indicated that leucines L43 and L112 within WD-repeats 1 and 2 respectively, are important for binding of PP2A Balpha to E4orf4. Furthermore, efforts to map the minimum sequence in PP2A Balpha required by E4orf4 were undertaken using truncation and internal deletion mutants. Small N-terminal deletions did not have an effect on the interaction of Balpha with E4orf4; however the binding reactions suggested that E4orf4 appears to interact with all 7 blades within the Balpha subunit. These studies suggested that although blades 1 and 2 may be critical for the interaction with E4orf4, association with the PP2A Balpha subunit may be complex.

Transcriptional regulation by nuclear receptors is vital for a number of different biological processes such as cellular differentiation, embryo development and homeostasis. Recent studies on the roles of estrogen-related receptor alpha (ERRalpha) and gamma (ERRgamma) have demonstrated an important function of these orphan receptors in the control of cardiac energy metabolism. Chromatin immunoprecipitation (ChIP)-on-chip was optimized and then performed using chromatin from mouse hearts on a genome-wide basis to provide further insight into the roles of these ERRs in the heart. In this thesis, we report the identification of ERRalpha and ERRgamma direct target genes in cardiac tissue implicated in various processes such as apoptosis, transcriptional regulation, signal transduction, heart development, electron transport/oxidative metabolism, as well as lipid, carbohydrate, and protein metabolism. However, one important and unexpected outcome of the experiments was the significant number of ERR target genes associated with cardiac hypertrophy in humans.

In Saccharomyces cerevisiae, zinc cluster proteins form a major family of transcriptional regulators for a variety of processes, yet many putative zinc cluster proteins have unknown functions. Previous studies assigned phenotypes to some of these proteins, a few of which showed previously unknown functions in pleiotropic drug resistance (PDR). The first study presented here focuses on a phenotypic analysis of double deletion mutants to further our understanding on functional relationships among zinc cluster proteins that mediate PDR. The second study focuses on a newly characterized zinc cluster protein, Asg1p, and its functional role in regulating stress response genes. In both studies, we found that the relationship among zinc cluster proteins is highly complex and tightly regulated.

The nature of protein-protein interactions was explored through the design of polypeptide ligands targeting specifically human a-thrombin. Design strategies ranged from isolation of binding fragments from natural proteins, conformational stabilization using structural scaffold, to bivalent linkage of library-selected components. / The interaction of thrombin with a 28-residue polypeptide from the sixth-epidermal growth factor-like repeat of human thrombomodulin (hTM-EGF6) was characterized in solution by use of NMR spectroscopy. The thrombin-binding region was identified and the thrombin-bound structure of the binding region was determined. The thrombin-bound structure of this fragment of thrombomodulin was then used as a basis for the design of peptide ligands with potentially enhanced thrombin-binding activities. Attempts for affinity enhancement through conformational stabilization by structure-based methods exemplified the need for alternative approaches that exploit the ubiquitous weak molecular interactions. / Bivalent ligands of thrombin were constructed via linkage of natural protein fragments. It was found that bivalent effects can be afforded by the proper linkage of the individually weak binding moieties. The bivalent designs were further generalized to include linkage of novel polypeptides selected from combinatorial libraries as individual binding components. These novel bivalent ligands targeting both the active site and the exosite I of thrombin exhibited up to 110-fold enhancement of binding activity. / The present study renews the interests in weak protein-protein/polypeptide interactions and their use in protein biochemistry. The design of thrombin-targeting bivalent ligands from weakly-binding moieties highlights the nature of bivalent molecular interactions, emphasizing the synergistic interplay between the two binding sites conferred by linker residues with a proper covalent geometry. It also illustrates that bivalent/multivalent binding can be applied as a general and practical approach for the design of high-affinity inhibitors targeting discrete sites on functionally important proteins, especially those involved in biological and cell signaling processes.

Previous studies strongly suggested that RBP1 regulated E2F-dependent transcription. A previous pilot experiment also suggested that RBP1 exited the nucleus during muscle cell differentiation. / In this study, repeated approaches and attempts consistently supported the alternative hypothesis that RBP1 remained nuclear during muscle differentiation. A key role for RBP1 in muscle differentiation was repeatedly suggested using RNAi. Specifically, siRNA was used to 'knock down' RBP1. The inability of myoblasts to form myotubes without RBP1 was illustrated by an inability of RBP1-depleted cells to express myosin after appropriate muscle-differention medium was applied. / Further initial work suggested that RBP1's probable essential role in muscle differentiation was independent of RBP1's role in regulating E2F-dependent transcription.

The Na,K-ATPase, or Na+ pump is an integral membrane protein found in the cells of virtually all higher eukaryotes and is one of the most important systems in cellular energy transduction. Na,K-ATPase catalyzes the electrogenic exchange of three intracellular Na+ for two extracellular K+ ions coupled to the hydrolysis of one molecule of ATP. The research described in this thesis concerns the regulation of the Na,K-ATPase by FXYD 2, a member of the FXYD family of small single transmembrane proteins. FXYD 2, commonly known as the gamma modulator, is located primarily in the kidney and has a role in modulating the enzyme's apparent affinities for ligands. This study has addressed several aspects of gamma structure and function, namely its function in intact cells, the function of the gamma transmembrane domain, and delineation of regions of the enzyme's catalytic alpha subunit with which gamma interacts. Transport assays using intact transfected HeLa cells showed that the two gamma variants, gammaa and gammab, cause (i) an increase in K+/Na+ antagonism, seen as an increase in K'Na at high K+ concentration, and (ii) an increase in apparent ATP affinity seen as an increase in ouabain-sensitive K+ influx as a function of ATP concentration. These results are consistent with those obtained earlier with unsided membrane preparations. The present study also showed a gamma-mediated increase in steady-state intracellular Na+ concentration and, in contrast to assays using permeabilized membranes, a gamma-mediated increase in apparent affinity for extracellular K+. Experiments with synthetic gamma transmembrane (gamma-TM) peptides provided insight into the role of the TM region such that incubation of these peptides with membranes containing alphabeta pumps modulated K'Na similarly to transfected full-length gamma, indicating that the TM domain alone can cause an increase in K'Na at high K+ concentration. Results with gamma-TM bearing the Gly41→Arg missense mutation associated with familial renal hypomagnesemia provided direct evidence that this mutation prevents gamma association with alphabeta pumps. In a study aimed to identify regions of alpha critical for the functional effects of gamma, interactions of alpha1/alpha2 (and the reverse alpha2/alpha1) chimeras with gamma showed the importance of the carboxy terminus, particularly TM 9. The chimera data also indicate that interactions of transmembrane regions of the catalytic alpha subunit with FXYD proteins are not necessarily the sole determinants of the kinetic effects of gamma on Na+ affinity since the extramembranous L7/8 loop of a appears to modulate intramembranous alpha-gamma interactions that mediated the increase in K+/Na+ antagonism.