Centrinas são proteínas componentes essenciais nos centro organizadores de microtubulos em diversos organismos. Pertencem à família das proteínas EF-Hand ligantes de cálcio e podem ser divididas em duas subfamílias: uma definida pela centrina da alga Chlamydomonas reinhardtii CrCenp, associada a funções contráteis, e a outra pela centrina da levedura Saccharomices cerevisiae ScCdc31p, relacionada a duplicação de centros mitóticos. Curiosamente o fungo Blastocladiella emersonii possui duas formas de centrinas em seu genoma : BeCen1 mais parecida com a CrCenp, e BeCen3 com a ScCdc31p, enquanto todos os outros fungos já descritos possuem apenas uma forma, pertencente ao grupo ScCdc31p. Diante desse fato curioso, descrito em 2005, estudos estruturais e termodinâmicos comparativos entre as proteínas recombinantes BeCen1 e BeCen3, foram desenvolvidos para identificar e caracterizar diferenças relevantes, que pudessem justificar a presença dessas duas proteínas no fungo Blatocladiella emersonii. Ambas as centrinas foram expressas em E. coli e purificadas por cromatografia, resultando em um rendimento de aproximadamente 5mg/l. Analises estruturais foram realizadas utilizando técnicas de dicroísmo circular (CD) , fluorescência, espalhamento de luz (DLS e RALS), microscopias de força atômica (AFM) e eletrônica de transmissão (MET). Por calorimetria de titulação isotérmica (ITC) parâmetros termodinâmicos foram obtidos para BeCen1 e BeCen3 em relação a ligação de cálcio. Ambas apresentaram conteúdo predominante de hélices alfa e diferenças físico-químicas e estruturais marcantes. BeCen1, após desnaturação a 90ºC e deixada a temperatura de 4°C por 24 horas horas, mostrou um espectro de CD compatível com um processo de reenovelamento; a TM foi de 42°C e aumentou cerca de 4°C na forma holo; os espectros de CD são modificados na presença de cálcio, mostrando uma forma característica de movimentação de hélices das proteínas ligantes de cálcio; pelos dados obtidos por ITC liga cálcio em três sítios EF-Hand por uma reação endotérmica; na presença de magnésio apresentou mudanças conformacionais, compatíveis com a ligação deste nos sítios de cálcio; a partir de 40°C é observado um processo de agregação chegando a formação de filamentos, que foram visualizados por AFM e MET. BeCen3 após ser desnaturada e colocada nas mesmas condições de BeCen1, permanece desnaturada; A TM é de 49°C e aumentou em aproximadamente 5°C na forma holo; os espectros de CD, na presença de cálcio, apresentam aspectos característicos de movimentação de -hélices das proteínas ligantes de cálcio; liga cálcio em apenas dois sítios EF-Hand por uma reação exotérmica, sofre mudanças conformacionais na presença de magnésio e a partir de 30°C já sofre um processo de agregação, formando filamentos. Neste trabalho foi estabelecido o primeiro protocolo para a expressão e purificação das centrinas de B. emersonii, além dos estudos de caracterização estrutural e termodinâmica destas proteínas. O estudo também foi pioneiro quanto a obtenção de imagens no processo de formação de filamentos destas centrinas na ausência de cálcio. As centrinas de B. emersonii apresentam diferenças importantes nas respostas em função da presença de cálcio e também do magnésio. Os dados obtidos são fortes indicativos que estas centrinas têm mecanismos de ação diferentes dentro do fungo B. emersonii. / Centrin proteins are essential component of the microtubule organizing centers in a large range of organisms. They belong to the EF-Hand calcium binding proteins family e can be divided in two subfamilies: one defined by the green algae Chlamydomonas reinhardtii CrCenp, related to contractile functions and the other centrin of the yeast Saccharomices cerevisiae ScCdc31p, related to duplication of centrossome mitotic centers. Interesantly, Blastocladiella emersonii fungus posses two centrin forms in it genome: BeCen1 closely related to CrCenp and BeCen3 closely related to ScCdc31p. All other known fungus have only one form of centrin: ScCdc31p. With this curious finding, described in 2005, compared structural and thermodynamics studies between recombinant proteins BeCen1 and BeCen3 were performed, in attempt to identify relevant differences that could explain the presence of two forms of centrins in this fungus. Both centrins were expressed in E. coli and purified by chromatography resulting in 5mg/l protein yield. Structural analyses were performed with circular dichroism (CD), fluorescence, light scattering (DLS AND RALS), atomic force microscopy (AFM) and transmission electronic microscopy (TEM). Using isothermal titration calorimetry (ITC) thermodynamics parameters about the calcium binding were defined. Both showed alpha helix predominant content and structural physical-chemistry differences. After denaturation at 90°C and cooled overnight at 4°C, BeCen1 showed a CD spectrum consistent to a renaturation process; the calculated TM was 42°C and raised 4°C in the holo state; CD spectra were modified under calcium presence showing characteristics changes of calcium binding proteins; ITC data exhibited tree calcium binding motifs through an endothermic reaction and the presence of magnesium also showed conformational changes; From 40°C a aggregation process leading to filament formation was observed and visualized with AFM and TEM. After denaturation at 90°C and cooled overnight at 4°C, BeCen3 remained denaturated; Calculated TM was 49°C and raised 5°C in the holo state; CD spectra were modified under calcium presence showing characteristics changes of calcium binding proteins; ITC data exhibited only two calcium binding motifs through an exothermic reaction and the presence of magnesium also showed conformational changes; From 30°C BeCen3 already suffered a aggregation process forming filaments. In this study it was established the first expression and purification protocol for B. emersonii centrins, besides the structural and thermodynamic characterization of these proteins. This is the first study containing filaments images of the centrins of B. emersonii. These centrins showed important response differences in calcium and magnesium binding. All the obtained data are strong indications that the two centrins have distinct functions in the fungus.

Centrinas são proteínas componentes essenciais nos centro organizadores de microtubulos em diversos organismos. Pertencem à família das proteínas EF-Hand ligantes de cálcio e podem ser divididas em duas subfamílias: uma definida pela centrina da alga Chlamydomonas reinhardtii CrCenp, associada a funções contráteis, e a outra pela centrina da levedura Saccharomices cerevisiae ScCdc31p, relacionada a duplicação de centros mitóticos. Curiosamente o fungo Blastocladiella emersonii possui duas formas de centrinas em seu genoma : BeCen1 mais parecida com a CrCenp, e BeCen3 com a ScCdc31p, enquanto todos os outros fungos já descritos possuem apenas uma forma, pertencente ao grupo ScCdc31p. Diante desse fato curioso, descrito em 2005, estudos estruturais e termodinâmicos comparativos entre as proteínas recombinantes BeCen1 e BeCen3, foram desenvolvidos para identificar e caracterizar diferenças relevantes, que pudessem justificar a presença dessas duas proteínas no fungo Blatocladiella emersonii. Ambas as centrinas foram expressas em E. coli e purificadas por cromatografia, resultando em um rendimento de aproximadamente 5mg/l. Analises estruturais foram realizadas utilizando técnicas de dicroísmo circular (CD) , fluorescência, espalhamento de luz (DLS e RALS), microscopias de força atômica (AFM) e eletrônica de transmissão (MET). Por calorimetria de titulação isotérmica (ITC) parâmetros termodinâmicos foram obtidos para BeCen1 e BeCen3 em relação a ligação de cálcio. Ambas apresentaram conteúdo predominante de hélices alfa e diferenças físico-químicas e estruturais marcantes. BeCen1, após desnaturação a 90ºC e deixada a temperatura de 4°C por 24 horas horas, mostrou um espectro de CD compatível com um processo de reenovelamento; a TM foi de 42°C e aumentou cerca de 4°C na forma holo; os espectros de CD são modificados na presença de cálcio, mostrando uma forma característica de movimentação de hélices das proteínas ligantes de cálcio; pelos dados obtidos por ITC liga cálcio em três sítios EF-Hand por uma reação endotérmica; na presença de magnésio apresentou mudanças conformacionais, compatíveis com a ligação deste nos sítios de cálcio; a partir de 40°C é observado um processo de agregação chegando a formação de filamentos, que foram visualizados por AFM e MET. BeCen3 após ser desnaturada e colocada nas mesmas condições de BeCen1, permanece desnaturada; A TM é de 49°C e aumentou em aproximadamente 5°C na forma holo; os espectros de CD, na presença de cálcio, apresentam aspectos característicos de movimentação de -hélices das proteínas ligantes de cálcio; liga cálcio em apenas dois sítios EF-Hand por uma reação exotérmica, sofre mudanças conformacionais na presença de magnésio e a partir de 30°C já sofre um processo de agregação, formando filamentos. Neste trabalho foi estabelecido o primeiro protocolo para a expressão e purificação das centrinas de B. emersonii, além dos estudos de caracterização estrutural e termodinâmica destas proteínas. O estudo também foi pioneiro quanto a obtenção de imagens no processo de formação de filamentos destas centrinas na ausência de cálcio. As centrinas de B. emersonii apresentam diferenças importantes nas respostas em função da presença de cálcio e também do magnésio. Os dados obtidos são fortes indicativos que estas centrinas têm mecanismos de ação diferentes dentro do fungo B. emersonii. / Centrin proteins are essential component of the microtubule organizing centers in a large range of organisms. They belong to the EF-Hand calcium binding proteins family e can be divided in two subfamilies: one defined by the green algae Chlamydomonas reinhardtii CrCenp, related to contractile functions and the other centrin of the yeast Saccharomices cerevisiae ScCdc31p, related to duplication of centrossome mitotic centers. Interesantly, Blastocladiella emersonii fungus posses two centrin forms in it genome: BeCen1 closely related to CrCenp and BeCen3 closely related to ScCdc31p. All other known fungus have only one form of centrin: ScCdc31p. With this curious finding, described in 2005, compared structural and thermodynamics studies between recombinant proteins BeCen1 and BeCen3 were performed, in attempt to identify relevant differences that could explain the presence of two forms of centrins in this fungus. Both centrins were expressed in E. coli and purified by chromatography resulting in 5mg/l protein yield. Structural analyses were performed with circular dichroism (CD), fluorescence, light scattering (DLS AND RALS), atomic force microscopy (AFM) and transmission electronic microscopy (TEM). Using isothermal titration calorimetry (ITC) thermodynamics parameters about the calcium binding were defined. Both showed alpha helix predominant content and structural physical-chemistry differences. After denaturation at 90°C and cooled overnight at 4°C, BeCen1 showed a CD spectrum consistent to a renaturation process; the calculated TM was 42°C and raised 4°C in the holo state; CD spectra were modified under calcium presence showing characteristics changes of calcium binding proteins; ITC data exhibited tree calcium binding motifs through an endothermic reaction and the presence of magnesium also showed conformational changes; From 40°C a aggregation process leading to filament formation was observed and visualized with AFM and TEM. After denaturation at 90°C and cooled overnight at 4°C, BeCen3 remained denaturated; Calculated TM was 49°C and raised 5°C in the holo state; CD spectra were modified under calcium presence showing characteristics changes of calcium binding proteins; ITC data exhibited only two calcium binding motifs through an exothermic reaction and the presence of magnesium also showed conformational changes; From 30°C BeCen3 already suffered a aggregation process forming filaments. In this study it was established the first expression and purification protocol for B. emersonii centrins, besides the structural and thermodynamic characterization of these proteins. This is the first study containing filaments images of the centrins of B. emersonii. These centrins showed important response differences in calcium and magnesium binding. All the obtained data are strong indications that the two centrins have distinct functions in the fungus.

Thesis advisor: Mary F. Roberts / Thesis advisor: Eranthie Weerapana / Peripheral proteins from mammals often exhibit multi-domain structures and require metal ions such as calcium as co-factors. This dissertation investigates two types of such proteins -- pulmonary collectins (surfactant proteins A and D) and phosphatidylinositol-specific phospholipase C (PLC) delta1 -- and their interactions with model membranes. One approach to work around the complexity brought upon by such multi-domain protein structure is to use a truncated construct or an isolated single domain. For pulmonary collectins, homotrimers consisting of the neck domain and the carbohydrate recognition domain were used in a novel NMR assay for better understanding of their lipid-specific interactions with the membranes. For PLC delta1, we were particularly interested in the role of the EF-hand domain. The isolated EF-hand domain of PLC delta1 was first used to characterize its interactions with membranes and identify key residues responsible for such interactions. These key residues in the N terminal lobe of the EF-hand domain, either cationic or hydrophobic, were then found to affect the hydrolysis activity of the full-length enzyme. A common role for this region of the PLC in facilitating proper membrane association was thus proposed. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Mon travail de doctorat a consisté à étudier in vitro la bioluminescence calciumdépendante de la photoprotéine aequorine puis d'utiliser cette bioluminescence afin deréaliser l'imagerie des activités du réseau néocortical. En effet, cette protéine peut être<br />exprimée dans des types cellulaires spécifiques et sa bioluminescence présente un rapportsignal/bruit très élevé et pas de toxicité.Dans un premier temps, j'ai recherché des mutations modifiant les propriétés debioluminescence de l'aequorine par une approche de mutagenèse aléatoire et d'évolution in vitro<br />J'ai ainsi obtenu des mutants dont la stabilité, la sensibilité calcique et/ou la cinétique debioluminescence étaient modifiées. L'étude de ces mutants a permis de mettre en évidence les relations étroites entre la cinétique d'émission de la bioluminescence et la<br />sensibilité calcique de l'aequorine. Ces travaux ont permis de mieux comprendre comment laliaison du calcium induit l'émission d'un photon par l'aequorine.Dans un second temps, j'ai développé une approche d'imagerie des activitésd'ensembles neuronaux grâce à une chimère GFP-aequorine exprimée en tranches denéocortex à l'aide d'un virus Sindbis recombinant. J'ai utilisé cette approche pour étudier lamodulation cholinergique de l'activité corticale évoquée par stimulation électrique. J'ai pumontrer que les agonistes muscariniques augmentent l'extension spatiale et la durée desréponses du réseau néocortical aux stimulations électriques.

Store-operated calcium entry (SOCE) is the major Ca2+ entry pathway in most non-excitable cells maintaining prolonged elevation of cytosolic Ca2+ levels required for gene transcription. SOCE is activated by the loss of endoplasmic reticulum (ER) Ca2+ through stromal interaction molecules (STIM), ER-membrane associated Ca2+ sensors. In humans, STIM1 and STIM2 share 65% sequence similarity but differentially regulate SOCE. Biophysical studies on the luminal Ca2+-binding region suggests that STIM2 EF-SAM is more stable than STIM1. The NMR structure of Ca2+-loaded STIM2 EF-SAM determined in this work suggests a more stable SAM and a tighter EF-hand and SAM interaction in STIM2 may be account for its higher stability. Chimeric swapping of the EF-hand and SAM domains generates an unstable ES211. Introducing ES211 into cherryFP-STIM1 shows constitutive puncta which activate SOCE independent of ER depletion. The current work demonstrates that the instability of the EF-SAM plays an important role in regulating SOCE initiation.

Store-operated calcium entry (SOCE) is the major Ca2+ entry pathway in most non-excitable cells maintaining prolonged elevation of cytosolic Ca2+ levels required for gene transcription. SOCE is activated by the loss of endoplasmic reticulum (ER) Ca2+ through stromal interaction molecules (STIM), ER-membrane associated Ca2+ sensors. In humans, STIM1 and STIM2 share 65% sequence similarity but differentially regulate SOCE. Biophysical studies on the luminal Ca2+-binding region suggests that STIM2 EF-SAM is more stable than STIM1. The NMR structure of Ca2+-loaded STIM2 EF-SAM determined in this work suggests a more stable SAM and a tighter EF-hand and SAM interaction in STIM2 may be account for its higher stability. Chimeric swapping of the EF-hand and SAM domains generates an unstable ES211. Introducing ES211 into cherryFP-STIM1 shows constitutive puncta which activate SOCE independent of ER depletion. The current work demonstrates that the instability of the EF-SAM plays an important role in regulating SOCE initiation.

Ca2+, a signal for death and life, is closely involved in the regulation of numerous important cellular events. Ca2+ carries out its function through its binding to Ca2+-receptors or Ca2+-binding proteins. The EF-hand protein, with a helix-loop-helix Ca2+-binding motif, constitutes one of the largest protein families. To facilitate our understanding of the role of Ca2+ in biological systems (denoted as calciomics) using genomic information, an improved pattern search method (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm) for the identification of EF-hand and EF-like Ca2+-binding proteins was developed. This fast and robust method allows us to analyze putative EF-hand proteins at the genome-wide level and further visualize the evolutionary scenario of the EF-hand protein family. This prediction method further enables us to locate a putative viral EF-hand Ca2+-binding motif within the rubella virus nonstructural protease that cleaves the nonstructural protein precursor into two active replicase components. A novel grafting approach has been used to probe the metal-binding properties of this motif by engineering the predicted 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2 domain 1. Structural and conformational studies were further performed on a purified, bacterially-expressed NS protease minimal metal-binding domain spanning the Zn2+- and EF-hand Ca2+-binding motif. It was revealed that Ca2+ binding induced local conformational changes and increased thermal stability. Furthermore, functional studies were carried out using RUB infectious cDNA clone and replicon constructs. Our studies have shown that the Ca2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. In addition, we have predicted and characterized a calmodulin-binding domain in the gap junction proteins connexin43 and connexin44. Peptides encompassing the CaM binding motifs were synthesized and their ability to bind CaM was determined using various biophysical approaches. Transient expression in HeLa cells of two mutant Cx43-EYFP constructs without the putative CaM-binding site eliminated the Ca2+-dependent inhibition of gap junction permeability. These results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 and Cx44 in a Ca2+-dependent manner, providing a molecular basis for the well-characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

Ca2+, both as a first and a second messenger, is closely involved in the modulation and regulation of numerous important cellular events, such as cell proliferation, differentiation and cell death. Fine-tuned Ca2+ signaling is achieved by its reversible or irreversible binding to a repertoire of Ca2+ signaling molecules. Among them, the extracellular calcium sensing receptor (CaSR) senses Ca2+ concentration ([Ca2+]o) in the milieu outside of cells where Ca2+ serves as a first messenger. An array of naturally-occurring mutations in CaSR has been found in patients with inherited disorders of Ca2+ homeostasis, leading to abnormal intracellular responses toward [Ca2+]o. In the present study, we have computationally predicted and experimentally characterized the metal-binding properties of five Ca2+-binding sites within CaSR and the accompanying metal--induced conformational changes by using two complementary methods-the grafting approach and the subdomain approach. Based on our results, a model has been proposed to explain the distinct CaSR-mediated responses toward abnormally ¡°high¡± or ¡°low¡± extracellular Ca2+ levels. In addition, we predicted and verified the interaction between CaSR with the most ubiquitously expressed four EF-hand-containing intracellular Ca2+ sensor protein, calmodulin (CaM). Our results demonstrate that the C-terminal CaM-binding domain of the CaSR is essential for proper intracellular Ca2+ response to external signals. Furthermore, we have applied the grafting approach to study the metal-binding properties and oligomeric state of the single EF-hand containing protein, STIM1. Our studies confirmed that the single EF-hand motif in STIM1, which resides in an equilibratium between its monomeric and dimeric forms, was capable of binding Ca2+ with a dissociation constant comparable to the ER Ca2+ concentration, suggesting it could function as a ER Ca2+ sensor responsible for sensing the Ca2+ filling state of ER.

Voltage-gated Cav2.1 (P/Q-type) and Cav2.2 (N-type) channels are two closely related calcium channels that play indispensable roles in signal transduction pathways by regulating neurotransmitter release. Despite having highly conserved amino acid sequences, they are differentially modulated by calmodulin, which mediate two important feedback mechanisms known as Ca2+-dependent inactivation (CDI) and Ca2+-dependent facilitation (CDF). These dual regulatory mechanisms contribute to synaptic plasticity, but only CDI is observed in Cav2.2 channel, while both CDI and CDF are present in Cav2.1 channel. Previously, it was hypothesized that the lack of CDF in Cav2.2 channel is due to the pre-IQ-IQ domain of the channel’s lower binding affinity for calmodulin compared to that of Cav2.1 channel. Now that the EF-hand domain of calcium channels is identified as one of the two minimally required molecular determinants that are responsible for supporting CDF in Cav2.1 channel and preventing CDF in Cav2.2 channel, it was necessary to determine the role of EF-hand domain in calmodulin binding of Cav2.1 and Cav2.2 channels. Using pull-down binding assays, this study finds that the EF-hand domain enhances calmodulin binding to the proximal C-terminal domain of Cav2.2 channel, which suggests that the lack of CDF in Cav2.2 does not result from the channel’s weak interaction with CaM, but from the EF-pre-IQ-IQ domain of the channel’s inability to allow calmodulin from fully exerting its effects.

The position of the cell division plane in plants is determined by the position of the preprophase band. The pre prophase band (PPB) is a ring of microtubules centered around the nucleus on the inner side of plasma membrane that establishes the cortical division site. The PPB forms at the end of G2 and breaks down at the end of prophase leaving behind protein markers of its position that are collectively called the cortical division site. During cytokinesis the phragmoplast expands towards the cortical division site and mediates the fusion of the new cell plate with the mother cell at that position. Several proteins necessary for PPB formation in plants have been identified, including maize DCD1 and ADD1 and Arabidopsis TON2, which are all type 2A protein phosphatase (PP2A)B" regulatory subunits. DCD1, ADD1, and TON2 localize to the PPB and the cortical division site through metaphase. The PP2A subunits each have two EF-hand domains, which are predicted to bind calcium ions. Since calcium ions are important for some aspects of cell division, we designed a series of constructs to test if TON2 binds calcium. TON2 protein was cloned into expression vectors, pET42a, and expression of TON2 protein was confirmed via Western blotting and immunodetection using a GST antibody. Site directed mutagenesis was used to mutate the TON2 EF-hand domains and mutated cDNAs were also cloned into expression vectors. These were then expressed in bacterial systems. Finally, the GST tagged proteins were purified. In the future, wild-type and mutated proteins TON2 proteins will used in calcium binding assays to determine if TON2 binds calcium.