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Le canal calcique dépendant du voltage de type-T CaV3.2 joue un rôle important dans l’excitabilité neuronale et dans la perception de la douleur. Le canal CaV3.2 partage une grande homologie structurale et fonctionnelle avec les canaux NaV. Ces deux types de canaux sont activés par de faibles dépolarisations membranaires et possèdent des cinétiques de temps d’activation et d’inactivation plus rapides que les canaux CaV de type-L. Les structures cristallines à haute résolution des canaux bactériens NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) et NaVRh (Zhang et al. 2012) suggèrent que l’hélice amphiphile S4S5 du domaine II peut être couplée avec les résidus de l’hélice S6 dans le domaine II ainsi qu’avec des résidus de l’hélice homologue dans le domaine adjacent, soit le domaine III, et ce, durant l’activation du canal. Pour déterminer les résidus fonctionnellement couplés, durant l’activation du canal CaV3.2, une analyse cyclique de doubles mutants a été effectuée par substitution en glycine et alanine des résidus clés entre l’hélice S4S5 du domaine II et le segment S6 des domaines II et III. Les propriétés biophysiques ont été mesurées à l’aide de la technique de « cut-open » sur les ovocytes. Les énergies d’activation ont été mesurées pour 47 mutations ponctuelles et pour 14 paires de mutants. De grandes énergies de couplage (ΔΔGinteract > 2 kcal mol-1) ont été observées pour 3 paires de mutants introduites dans les IIS4S5/IIS6 et IIS4S5/IIIS6. Aucun couplage significatif n’a été observé entre le IIS4S5 et le IVS6. Nos résultats semblent démontrer que les hélices S4S5 et S6 provenant de deux domaines voisins sont couplées durant l’activation du canal calcique de type-T CaV3.2. / Voltage-activated T-type calcium channel CaV3.2 plays an important role in neuronal excitability and in pain perception. CaV3.2 channel bears a strong structural and functional homology with voltage-dependent NaV channels. In particular, these channels are activated by relatively small depolarization and display faster activation and inactivation kinetics than the L-type CaV channel. High-resolution crystal structures of bacterial NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) and NaVRh (Zhang et al. 2012) suggest that the amphiphilic helix S4S5 in Domain II may be coupled with S6 residues both in Domain II and in the adjacent Domain III during channel activation.To determine whether residues in the S4S5 helix of Domain II are functionally coupled with residues in the S6 helix in Domain II and Domain III during the voltage-dependent activation of CaV3.2, a double mutant cycle analysis was performed by introducing pairs of glycine and alanine residues in the S4S5 helix of Domain II and the S6 region of Domains II and III. Biophysical properties were measured with the cut-open oocyte technique. Activation gating was measured for 47 single mutants and 14 pairs of mutants. Strong coupling energies (ΔΔGinteract > 2 kcal mol-1) were reported for 3 pairs of mutants introduced in IIS4S5/IIS6 and IIS4S5/IIIS6. No significant coupling was observed between IIS4S5 and IVS6. Altogether, our results demonstrate that the S4S5 and S6 helices from neighboring domains are energetically coupled during the activation of the low voltage-gated T-type CaV3.2 channel.

Le canal calcique dépendant du voltage de type-T CaV3.2 joue un rôle important dans l’excitabilité neuronale et dans la perception de la douleur. Le canal CaV3.2 partage une grande homologie structurale et fonctionnelle avec les canaux NaV. Ces deux types de canaux sont activés par de faibles dépolarisations membranaires et possèdent des cinétiques de temps d’activation et d’inactivation plus rapides que les canaux CaV de type-L. Les structures cristallines à haute résolution des canaux bactériens NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) et NaVRh (Zhang et al. 2012) suggèrent que l’hélice amphiphile S4S5 du domaine II peut être couplée avec les résidus de l’hélice S6 dans le domaine II ainsi qu’avec des résidus de l’hélice homologue dans le domaine adjacent, soit le domaine III, et ce, durant l’activation du canal. Pour déterminer les résidus fonctionnellement couplés, durant l’activation du canal CaV3.2, une analyse cyclique de doubles mutants a été effectuée par substitution en glycine et alanine des résidus clés entre l’hélice S4S5 du domaine II et le segment S6 des domaines II et III. Les propriétés biophysiques ont été mesurées à l’aide de la technique de « cut-open » sur les ovocytes. Les énergies d’activation ont été mesurées pour 47 mutations ponctuelles et pour 14 paires de mutants. De grandes énergies de couplage (ΔΔGinteract > 2 kcal mol-1) ont été observées pour 3 paires de mutants introduites dans les IIS4S5/IIS6 et IIS4S5/IIIS6. Aucun couplage significatif n’a été observé entre le IIS4S5 et le IVS6. Nos résultats semblent démontrer que les hélices S4S5 et S6 provenant de deux domaines voisins sont couplées durant l’activation du canal calcique de type-T CaV3.2. / Voltage-activated T-type calcium channel CaV3.2 plays an important role in neuronal excitability and in pain perception. CaV3.2 channel bears a strong structural and functional homology with voltage-dependent NaV channels. In particular, these channels are activated by relatively small depolarization and display faster activation and inactivation kinetics than the L-type CaV channel. High-resolution crystal structures of bacterial NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) and NaVRh (Zhang et al. 2012) suggest that the amphiphilic helix S4S5 in Domain II may be coupled with S6 residues both in Domain II and in the adjacent Domain III during channel activation.To determine whether residues in the S4S5 helix of Domain II are functionally coupled with residues in the S6 helix in Domain II and Domain III during the voltage-dependent activation of CaV3.2, a double mutant cycle analysis was performed by introducing pairs of glycine and alanine residues in the S4S5 helix of Domain II and the S6 region of Domains II and III. Biophysical properties were measured with the cut-open oocyte technique. Activation gating was measured for 47 single mutants and 14 pairs of mutants. Strong coupling energies (ΔΔGinteract > 2 kcal mol-1) were reported for 3 pairs of mutants introduced in IIS4S5/IIS6 and IIS4S5/IIIS6. No significant coupling was observed between IIS4S5 and IVS6. Altogether, our results demonstrate that the S4S5 and S6 helices from neighboring domains are energetically coupled during the activation of the low voltage-gated T-type CaV3.2 channel.

Coiled coils are a well-known protein structure prevalent in eukaryotic function, synthetic applications, and de novo protein design. Coiled-coil folding is often described using heptad repeat positions labeled abcdefg where a and d positions occupy the interface between the coils, e and g positions flank the interface, and the b, c, and f positions face the solvent-exposed surface. The a, d, e, and g positions have been extensively studied in the coiled-coil literature. There is a lack of investigation on the impact of the b, c, and f positions on coiled-coil folding. Chapter 1 is an introduction to the heptad repeat of coiled coils and the impact on folding of each heptad repeat position. In Chapter 2 we introduce a non-covalent interaction among the b, c, and f positions of a coiled-coil trimer that significantly enhances thermodynamic stability. We identify characteristics of the f-position residue (hydrogen bond donating ability and hydrophobicity) that lead to the greatest amount of stability. Chapter 3 introduces crystal structures and molecular dynamic simulations of the interaction to identify the mechanism of stabilization. Further thermodynamic studies find a key salt-bridge interaction between the b and c positions that are influenced by the f-position residue. Chapter 4 explores the impact of salt on the non-covalent interaction and determines that the interaction is sensitive to salt screening and is ionic in nature. It also explores more characteristics of the f-position amino acid, in particular the hydrogen bond donating component. In Chapter 5 we insert the solvent-exposed interaction into helix bundles of differing length and oligomeric state. We find that stability is not only dependent upon amino acid identity but also the length and stoichiometry of a coiled coil.

Les canaux calciques dépendants du voltage CaV font partie de la famille structurale des canaux ioniques à 6 segments transmembranaires. Tout comme les canaux potassiques Kv, les canaux CaV possèdent une série de résidus chargés dans l’hélice S4 de chaque domaine ou sous-unité qui conférerait à la protéine une sensibilité aux changements de voltage. De plus les hélices S6 tapissent la paroi du pore et forment la porte d’activation de la protéine. Comment le mouvement des hélices S4 se traduit par l’ouverture de la porte d’activation des hélices S6 demeure une question encore non résolue. Suite à la publication de la structure cristalline du canal Kv1.2 en 2005, le groupe de MacKinnon a proposé que le mouvement des hélices S4 est mécaniquement couplé à la porte d’activation S6 à travers le glissement de l’hélice amphiphile S4-S5 selon un mécanisme nommé couplage électromécanique (Long et al. 2005b). Dans le but de déterminer si la région S4-S5 joue un rôle dans l’activation du canal calcique CaV2.3, nous avons étudié, par la méthode d’analyse cyclique de mutations doubles (« Double Mutant Cycle Analysis », (Horovitz 1996)), le couplage entre la boucle S4-S5 et l’hélice S6 du domaine II de ce canal. Les mesures d’énergies d’activation, ΔGact, obtenues en présence des sous-unités auxiliaires CaVα2δ et CaVβ3 ont affiché un couplage significatif pour l’activation entre les paires de résidus V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, et S602G/I701G. Aucune de ces paires de résidus n’a affiché de couplage lors de l’inactivation, suggérant que les effets observés sont spécifiques au mécanisme d’activation. Mis ensemble, ces résultats suggèrent que la boucle IIS4-S5 et l’hélice IIS6 interagissent et jouent un rôle déterminant dans l’activation de CaV2.3. / Voltage dependent calcium channels share a strong structural homology with voltage gated potassium channels. Both families present a conserved series of charged residues present in the S4 helix of each domain that most certainly accounts for the voltage sensitivity of these proteins. Moreover, in both cases, the S6 helices seem to be lining up the pore. How does the movement of the S4 sensors translate into channel opening remains elusive in Ca2+ channels. Following the publication of the crystal structure of the Kv1.2 channel in 2005, the group of Roderick MacKinnon proposed that the voltage sensor is mechanically coupled to the S6 pore through the amphipathic S4-S5 helix that crosses over the S6 inner helix from the same subunit. To determine if the S4-S5 linker, that runs parallel to the membrane plane inside the cell in the Kv1.2 three-D structure, plays a role in the activation of the CaV2.3 calcium channel, we have studied by double mutant cycle analysis the coupling between the S4-S5 linker and the S6 helix of domain II of this channel. The activation energies, Gact, obtained from classical two electrode voltage clamp experiments in the presence of auxiliary subunits CaV2 and CaV3 displayed significant activation coupling coefficients for the pairs of residues V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, and S602G/I701G. None of these pairs displayed significant coupling in the inactivation mechanism, suggesting that the effects observed were specific to activation. Altogether, our results strongly suggest that the S4-S5 linker and the S6 helix of domain II are actively involved in the activation of CaV2.3.

Tropomiosina (Tm) é uma das proteínas que compõe o filamento fino (actina, Tm, Troponina) do sistema muscular esquelético e desempenha um importante papel na regulação da contração muscular. Tm é um coiled-coil de 284 resíduos que forma longos homopolímeros lineares através da sobreposição de onze resíduos entre os terminais de Tms adjacentes (Interação cabeça-cauda) em condições de baixa força iônica. A presença de vários resíduos carregados (D2, K5, K6, K7, D275, H276 e D280) nas extremidades da Tm sugere que contatos intermoleculares eletrostáticos entre estes aminoácidos podem ter um importante papel na estabilidade dos polímeros. Entretanto, a estrutura do complexo cabeça-cauda demonstra que a maioria dos contatos intermoleculares na interface é de natureza hidrofóbica. A fim de analisarmos a contribuição dos grupos carregados para a estabilidade do complexo cabeça-cauda, construímos fragmentos recombinantes correspondentes à metade amino (ASTm1-142 ) e carboxi (Tm143-284(5OHW269)) terminais da proteína contendo mutações pontuais daqueles resíduos para alanina, e adicionalmente H276 para Glu. Medimos a afinidade entre todas as possíveis combinações destes fragmentos na ausência e presença de íons Mg2+, visto que este cátion está sempre presente em condições fisiológicas e é importante para estabilizar a interação entre Tm e actina. Os efeitos das mutações foram analisados por simulações de docking, desnaturações térmicas e ciclos de duplos mutantes. Os resultados demonstram que os aminoácidos K5, K7 e D280 presentes na interface formam contatos intermoleculares essenciais para a estabilidade do complexo. Enquanto, D2, K6, D275 e H276 não participam na formação de contatos intermoleculares, no entanto, contribuem para a estabilidade da interação cabeça- cauda através de suas interações intramoleculares que atuam na estabilidade das hélices individuais. Os aumentos na estabilidade da metade C-terminal da Tm (Tm143-284(5OHW)) induzidos por Mg2+ foram dependentes das mutações neste trecho da proteína sugerindo a presença de um sítio de ligação para este íon na extremidade carboxi terminal da molécula no trecho que forma a interação cabeça- cauda. Construímos um fragmento menor do C-terminal (Tm259-284(W269)) para acompanharmos mudanças no deslocamento químico induzidas pela ligação do íon usando ressonância magnética nuclear. Os resultados obtidos comprovaram nossa hipótese e nos permitiram definir pela primeira vez que a estrutura da Tm tem um ou mais sítios de ligação Mg2+ em uma região próxima ao resíduo H276 que está localizado entre vários resíduos carregados negativamente que participam da interação cabeça-cauda. Por último, estudamos os efeitos de solventes cosmótropicos (TFE e glicerol) nas estabilidades dos fragmentos da Tm, uma vez que a instabilidade (flexibilidade) da extremidade C-terminal é importante para a formação do complexo cabeça-cauda. Observamos que TFE, porém não glicerol, reduziu a afinidade entre os terminais. Ambos os co-solventes induziram aumentos na estabilidade dos fragmentos, no entanto, apenas TFE induziu um aumento no conteúdo de α-hélice e causou uma redução significativa na cooperatividade de desenovelamento das proteínas. Estes resultados indicam que estes compostos orgânicos estabilizam as estruturas dos fragmentos individuais da Tm de maneiras diferentes e que estas diferenças podem estar relacionadas aos diferentes efeitos observados na formação da interação cabeça-cauda. / Tropomyosin (Tm) is a protein component of the skeletal muscle thin filament (actin, Tm, Troponin) which has an important role in the regulation of muscle contraction. Tm is a dimeric coiled-coil (284 aminoacids) which forms long linear homopolymers through the overlap of eleven residues of adjacent Tm termini (Head- to-tail interaction) in low ionic strength conditions. The presence of several charged amino acids (D2, K5, K6, K7, D275, H276 e D280) in Tm extremities suggests that electrostatic contacts among those residues may have an important role in the stability of the polymers. Nevertheless, the solution structure of the head-to-tail complex demonstrated that most of the contacts in the interface are hydrophobic. In order to study the contribution of these charged residues to the stability of the head- to-tail complex, we built recombinant fragments corresponding to the amino (ASTm1-142) and carboxy (Tm143-284(5OHW269)) termini containing single mutations of those amino acids to alanine, and additionally a substitution of H276 for Glu. We measured the binding affinities among all possible combinations of wild-type and mutant fragments in the absence or presence of Mg2+ ions. This cation is always physiologically present in the muscle and it is known to strengthen the binding of Tm to actin. The effects of the mutations were analyzed by protein-protein docking, thermodynamic cycles and thermal denaturations. The results show that residues K5, K7 and D280 are essential to the stability of the complex. Though D2, K6, D275 and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to the complex stability by modulating the stability of the helices at the Tm termini. Mg2+-induced increases in stability of the C- terminal were sensitive to mutations in residues located in the head-to-tail overlap region, suggesting that Mg2+ ions may bind specifically to the carboxy extremity of the protein. We produced a small peptide (Tm259-284(W269)) to follow amide chemical shift perturbations upon Mg2+ binding by nuclear magnetic resonance measurements. The results obtained with this peptide allowed us to define for the first time that the Tm structure has one or more Mg2+ binding sites in a region centered in the vicinity of H276 in which are located several negatively charged residues that participate in the head-to-tail interaction. We also studied the effects of kosmotropic co-solvents (TFE and glycerol) in the stability of Tm fragments, as the instability (flexibility) of the C- terminal region has been pointed as important for the formation of the head-to-tail complex. We observed that TFE, but not glycerol, reduces the affinity between the termini. Both TFE and glycerol increased the stability of the isolated N- and C- terminal fragments; however, only TFE caused an increase in the helical content and a significant reduction in the cooperativity of unfolding of the proteins. Our results show that these two co-solvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their different effects on head-to-tail complex formation.

Tropomiosina (Tm) é uma das proteínas que compõe o filamento fino (actina, Tm, Troponina) do sistema muscular esquelético e desempenha um importante papel na regulação da contração muscular. Tm é um coiled-coil de 284 resíduos que forma longos homopolímeros lineares através da sobreposição de onze resíduos entre os terminais de Tms adjacentes (Interação cabeça-cauda) em condições de baixa força iônica. A presença de vários resíduos carregados (D2, K5, K6, K7, D275, H276 e D280) nas extremidades da Tm sugere que contatos intermoleculares eletrostáticos entre estes aminoácidos podem ter um importante papel na estabilidade dos polímeros. Entretanto, a estrutura do complexo cabeça-cauda demonstra que a maioria dos contatos intermoleculares na interface é de natureza hidrofóbica. A fim de analisarmos a contribuição dos grupos carregados para a estabilidade do complexo cabeça-cauda, construímos fragmentos recombinantes correspondentes à metade amino (ASTm1-142 ) e carboxi (Tm143-284(5OHW269)) terminais da proteína contendo mutações pontuais daqueles resíduos para alanina, e adicionalmente H276 para Glu. Medimos a afinidade entre todas as possíveis combinações destes fragmentos na ausência e presença de íons Mg2+, visto que este cátion está sempre presente em condições fisiológicas e é importante para estabilizar a interação entre Tm e actina. Os efeitos das mutações foram analisados por simulações de docking, desnaturações térmicas e ciclos de duplos mutantes. Os resultados demonstram que os aminoácidos K5, K7 e D280 presentes na interface formam contatos intermoleculares essenciais para a estabilidade do complexo. Enquanto, D2, K6, D275 e H276 não participam na formação de contatos intermoleculares, no entanto, contribuem para a estabilidade da interação cabeça- cauda através de suas interações intramoleculares que atuam na estabilidade das hélices individuais. Os aumentos na estabilidade da metade C-terminal da Tm (Tm143-284(5OHW)) induzidos por Mg2+ foram dependentes das mutações neste trecho da proteína sugerindo a presença de um sítio de ligação para este íon na extremidade carboxi terminal da molécula no trecho que forma a interação cabeça- cauda. Construímos um fragmento menor do C-terminal (Tm259-284(W269)) para acompanharmos mudanças no deslocamento químico induzidas pela ligação do íon usando ressonância magnética nuclear. Os resultados obtidos comprovaram nossa hipótese e nos permitiram definir pela primeira vez que a estrutura da Tm tem um ou mais sítios de ligação Mg2+ em uma região próxima ao resíduo H276 que está localizado entre vários resíduos carregados negativamente que participam da interação cabeça-cauda. Por último, estudamos os efeitos de solventes cosmótropicos (TFE e glicerol) nas estabilidades dos fragmentos da Tm, uma vez que a instabilidade (flexibilidade) da extremidade C-terminal é importante para a formação do complexo cabeça-cauda. Observamos que TFE, porém não glicerol, reduziu a afinidade entre os terminais. Ambos os co-solventes induziram aumentos na estabilidade dos fragmentos, no entanto, apenas TFE induziu um aumento no conteúdo de α-hélice e causou uma redução significativa na cooperatividade de desenovelamento das proteínas. Estes resultados indicam que estes compostos orgânicos estabilizam as estruturas dos fragmentos individuais da Tm de maneiras diferentes e que estas diferenças podem estar relacionadas aos diferentes efeitos observados na formação da interação cabeça-cauda. / Tropomyosin (Tm) is a protein component of the skeletal muscle thin filament (actin, Tm, Troponin) which has an important role in the regulation of muscle contraction. Tm is a dimeric coiled-coil (284 aminoacids) which forms long linear homopolymers through the overlap of eleven residues of adjacent Tm termini (Head- to-tail interaction) in low ionic strength conditions. The presence of several charged amino acids (D2, K5, K6, K7, D275, H276 e D280) in Tm extremities suggests that electrostatic contacts among those residues may have an important role in the stability of the polymers. Nevertheless, the solution structure of the head-to-tail complex demonstrated that most of the contacts in the interface are hydrophobic. In order to study the contribution of these charged residues to the stability of the head- to-tail complex, we built recombinant fragments corresponding to the amino (ASTm1-142) and carboxy (Tm143-284(5OHW269)) termini containing single mutations of those amino acids to alanine, and additionally a substitution of H276 for Glu. We measured the binding affinities among all possible combinations of wild-type and mutant fragments in the absence or presence of Mg2+ ions. This cation is always physiologically present in the muscle and it is known to strengthen the binding of Tm to actin. The effects of the mutations were analyzed by protein-protein docking, thermodynamic cycles and thermal denaturations. The results show that residues K5, K7 and D280 are essential to the stability of the complex. Though D2, K6, D275 and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to the complex stability by modulating the stability of the helices at the Tm termini. Mg2+-induced increases in stability of the C- terminal were sensitive to mutations in residues located in the head-to-tail overlap region, suggesting that Mg2+ ions may bind specifically to the carboxy extremity of the protein. We produced a small peptide (Tm259-284(W269)) to follow amide chemical shift perturbations upon Mg2+ binding by nuclear magnetic resonance measurements. The results obtained with this peptide allowed us to define for the first time that the Tm structure has one or more Mg2+ binding sites in a region centered in the vicinity of H276 in which are located several negatively charged residues that participate in the head-to-tail interaction. We also studied the effects of kosmotropic co-solvents (TFE and glycerol) in the stability of Tm fragments, as the instability (flexibility) of the C- terminal region has been pointed as important for the formation of the head-to-tail complex. We observed that TFE, but not glycerol, reduces the affinity between the termini. Both TFE and glycerol increased the stability of the isolated N- and C- terminal fragments; however, only TFE caused an increase in the helical content and a significant reduction in the cooperativity of unfolding of the proteins. Our results show that these two co-solvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their different effects on head-to-tail complex formation.

Les canaux calciques dépendants du voltage CaV font partie de la famille structurale des canaux ioniques à 6 segments transmembranaires. Tout comme les canaux potassiques Kv, les canaux CaV possèdent une série de résidus chargés dans l’hélice S4 de chaque domaine ou sous-unité qui conférerait à la protéine une sensibilité aux changements de voltage. De plus les hélices S6 tapissent la paroi du pore et forment la porte d’activation de la protéine. Comment le mouvement des hélices S4 se traduit par l’ouverture de la porte d’activation des hélices S6 demeure une question encore non résolue. Suite à la publication de la structure cristalline du canal Kv1.2 en 2005, le groupe de MacKinnon a proposé que le mouvement des hélices S4 est mécaniquement couplé à la porte d’activation S6 à travers le glissement de l’hélice amphiphile S4-S5 selon un mécanisme nommé couplage électromécanique (Long et al. 2005b). Dans le but de déterminer si la région S4-S5 joue un rôle dans l’activation du canal calcique CaV2.3, nous avons étudié, par la méthode d’analyse cyclique de mutations doubles (« Double Mutant Cycle Analysis », (Horovitz 1996)), le couplage entre la boucle S4-S5 et l’hélice S6 du domaine II de ce canal. Les mesures d’énergies d’activation, ΔGact, obtenues en présence des sous-unités auxiliaires CaVα2δ et CaVβ3 ont affiché un couplage significatif pour l’activation entre les paires de résidus V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, et S602G/I701G. Aucune de ces paires de résidus n’a affiché de couplage lors de l’inactivation, suggérant que les effets observés sont spécifiques au mécanisme d’activation. Mis ensemble, ces résultats suggèrent que la boucle IIS4-S5 et l’hélice IIS6 interagissent et jouent un rôle déterminant dans l’activation de CaV2.3. / Voltage dependent calcium channels share a strong structural homology with voltage gated potassium channels. Both families present a conserved series of charged residues present in the S4 helix of each domain that most certainly accounts for the voltage sensitivity of these proteins. Moreover, in both cases, the S6 helices seem to be lining up the pore. How does the movement of the S4 sensors translate into channel opening remains elusive in Ca2+ channels. Following the publication of the crystal structure of the Kv1.2 channel in 2005, the group of Roderick MacKinnon proposed that the voltage sensor is mechanically coupled to the S6 pore through the amphipathic S4-S5 helix that crosses over the S6 inner helix from the same subunit. To determine if the S4-S5 linker, that runs parallel to the membrane plane inside the cell in the Kv1.2 three-D structure, plays a role in the activation of the CaV2.3 calcium channel, we have studied by double mutant cycle analysis the coupling between the S4-S5 linker and the S6 helix of domain II of this channel. The activation energies, Gact, obtained from classical two electrode voltage clamp experiments in the presence of auxiliary subunits CaV2 and CaV3 displayed significant activation coupling coefficients for the pairs of residues V593G/L699G, V593G/A700G, V593G/A702G, S595G/V703G L596G/L699G, L596G/A700G, L596G/I701G, L596G/A702G, L596G/V703G, L596G/D704G, M597G/I701G, and S602G/I701G. None of these pairs displayed significant coupling in the inactivation mechanism, suggesting that the effects observed were specific to activation. Altogether, our results strongly suggest that the S4-S5 linker and the S6 helix of domain II are actively involved in the activation of CaV2.3.

<p>Early processes following photon absorption by the photosynthetic pigment-protein complex photosystem I (PS I) have been the subject of decades of research, yet many questions remain in this area of study. Among the trickiest to investigate is the role of the PS I reaction center’s (RC’s) two accessory (A_‑1) chlorophyll (Chl) cofactors as primary electron donors or acceptors, oxidizing the special pair (P₇₀₀) of Chls or reducing a nominal primary electron acceptor (A₀) Chl in the first electron transfer step. Such processes, which occur on a picosecond timescale, have long been studied via ultrafast spectroscopy, though difficulty lies in distinguishing among signals from early processes, which have similar lifetimes and involve many identical pigments. In this work, we used steady-state and ultrafast optical pump-probe spectroscopies on PS I trimers from wildtype and mutant strains of the cyanobacterium <i>Synechocystis</i> sp. PCC 6803 in which an asparagine amino acid residue near A_‑1 had been replaced with methionine on one or both sides of the RC. We also conducted an identical set of experiments on mutants in which A₀ was similarly targeted, as well as studied the effects on the A₀ absorption spectrum of a third category of mutations in which a peripheral H‑bond to A₀ was lost. Steady-state absorption spectroscopy revealed that many of these mutations caused mild Chl deficiencies in the light-capturing antenna of PS I without necessarily preventing organisms’ growth. More importantly, we determined that contrary to certain hypotheses, A_‑1 is the most likely true first electron acceptor, as reasoned from observing rapid triplet state formation in double A_‑1 mutants. We also concluded from non-additive detrimental effects of single-side mutations that if one RC branch is damaged at the level of A₀ or A_‑1, electron transfer may be redirected along the intact branch. This may help explain the conservation of two functional RC branches in PS I over many generations of natural selection, despite the additional cost to organisms of manufacturing both.</p>

The ability of plants to sense and respond to environmental and endogenous signals is essential to their growth and development. As part of these diverse cellular functions, ubiquitin-mediated proteolysis has emerged to be an important process involved in how plant signalling pathways can be regulated in response to such cues. Of the three enzymes involved in linking ubiquitin to protein targets, E3 ubiquitin ligases are of interest as they confer substrate specificity during this ubiquitination process. The overall focal point of this research is on plant U-box (PUB) E3 ubiquitin ligases, a family that has undergone a large gene expansion possibly attributable to the regulation of biological processes unique to the plant life cycle. In Arabidopsis there are 64 predicted PUBs, many for which biological roles have yet to be determined. And as research continues to uncover PUB functions, the functional diversity in the gene family will likely expand. Specifically the focus of this research is on characterizing two ARM repeat-containing PUBs – AtPUB18 and AtPUB19. General analysis of pub18 and pub19 T-DNA insertion lines for growth defects did not yield distinct altered phenotypes. Closer inspection of selected lines showed independent gene assortment phenotypes that, with further inordinately convoluted pursuit, proved to have an AtPUB18/19-unrelated outcome. The availability of Arabidopsis microarray databases provided exploratory expression profiling as a starting point to elucidate PUB function. AtPUB19 and closely related AtPUB18 are notable for their increased expression during abiotic stresses. While condition-directed germination assays showed a decreased sensitivity to salt and ABA for pub18 pub19 double insertion lines, no related change in susceptibility to these or other abiotic stress treatments were seen with condition-directed root growth assays. Thus, this preliminary work has begun to reveal insight into the complex abiotic stress-related roles AtPUB18 and AtPUB19 have during mediation of environmental stress acclimation in Arabidopsis.