The crystal structure of a biological insulated transmembrane molecular wire

A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand β-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes

Characterisation of MtoD from Sideroxydans lithotrophicus: a cytochrome c electron shuttle used in lithoautotrophic growth

The autotrophic Sideroxydans lithotrophicus ES-1 can grow by coupling the oxidation of ferrous iron to the reduction of oxygen. Soluble ferrous iron is oxidised at the surface of the cell by an MtoAB porin-cytochrome complex that functions as an electron conduit through the outer membrane. Electrons are then transported to the cytoplasmic membrane where they are used to generate proton motive force (for ATP synthesis) and NADH for autotrophic processes such as carbon fixation. As part of the mtoAB gene cluster, S. lithotrophicus also contains the gene mtoD that is proposed to encode a cytochrome c protein. We isolated mtoD from a Shewanella oneidensis expression system where the mtoD gene was expressed on a pBAD plasmid vector. Biochemical, biophysical and crystallographic characterisation of the purified MtoD revealed it as an 11 kDa monomeric protein containing a single heme. Sequence and structural alignment indicated that MtoD belonged to the class-1 cytochrome c family and had a similar fold to ferricytochrome c552 family, however the MtoD heme is bis-histidine coordinated and is substantially more exposed than the hemes of other family members. The reduction potential of the MtoD heme at pH 7 was +155 mV vs. Standard Hydrogen Electrode, which is approximately 100 mV lower than that of mitochondrial cytochromes c. Consideration of the properties of MtoD in the context of the potential respiratory partners identified from the genome suggests that MtoD could associate to multiple electron transfer partners as the primary periplasmic electron shuttle

Novel partners support two-way by-product mutualism in a converted ecosystem

Dissertação de Mestrado em Química Medicinal apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.Nas sociedades modernas assiste-se a um aumento crescente do número de bactérias e vírus com resistência aos fármacos atualmente disponibilizados pela indústria farmacêutica. Dada a relevância da problemática mencionada, o objetivo central do trabalho apresentado nesta dissertação consiste no desenvolvimento de métodos de síntese de novas entidades químicas biocompatíveis constituídas por macrociclos tetrapirrólicos e sulfonamidas, tendo em vista a sua futura potencial aplicação dual como antibacteriano per si e como fotossensibilizador para inativação de bactérias recorrendo a terapia fotodinâmica. A combinação destas duas famílias de compostos pode ocorrer por ligação covalente ou via self-assembly, subdividindo o trabalho apresentado. No capítulo 1 apresenta-se uma revisão crítica e selecionada da literatura subjacente aos diferentes tópicos estudados. No capítulo 2 descreve-se uma estratégia de síntese de macrociclos tetrapirrólicos e sulfonamidas unidas por ligação covalente. Neste começa-se por selecionar a a 5,10,15,20-tetraquis(pentafluorofenil)porfirina como porfirina base para realizar os estudos de otimização da sua derivatização com a metanosulfonamida, conseguindo-se obter com sucesso condições de síntese seletivas para a formação da 5-[2’,3’,5’,6’-tetrafluoro-4’- metanosulfamoil)fenil]-10,15,20-tri-[(2’,3’,4’,5’,6’-pentafluoro)fenil]porfirina (composto monossubstituido) ou da 5,10,15,20-tetra-[2’,3’,5’,6’-tetrafluoro-4’-metanosulfamoil)fenil]porfirina (composto tetrassubstituído) com rendimentos de 19% e 70%, respetivamente. Com o intuito de avaliar o efeito da estrutura da sulfonamida na reatividade alargaram-se os estudos utilizando como nucleófilo duas sulfonamidas previamente sintetizadas no decorrer do trabalho: ptoluenosulfonamida e N-metil-p-toluenosulfonamida. Através deste estudo foi possível sintetizar e isolar os compostos mono e dissubstituídos da família da p-toluenosulfonamida (5-[(2’,3’,5’,6’- tetrafluoro-4’-p-toluenosulfamoil)fenil]-10,15,20-tri-[(2’,3’,4’,5’,6’-pentafluoro)fenil]porfirina e mistura de 5,10-[(2’,3’,5’,6’-tetrafluoro-4’-p-toluenosulfamoil)fenil]-15,20-dis-[(2’,3’,4’,5’,6’- pentafluoro)fenil]porfirina e 5,15-[(2’,3’,5’,6’-tetrafluoro-4’-p-toluenosulfamoil)fenil]-10,20- [(2’,3’,4’,5’,6’-pentafluoro)fenil]porfirina) e os compostos mono, di e trissubstituído referentes à substituição com a N-metil-p-toluenosulfonamida (5-[(2’,3’,5’,6’-tetrafluoro-4’-N-metil-ptoluenosulfamoil) fenil]-10,15,20-tri-[(2’,3’,4’,5’,6’-pentafluoro)fenil] porfirina, mistura de 5,10- [(2’,3’,5’,6’-tetrafluoro-4’-N-metil-p-toluenosulfamoil)fenil]-15,20-dis-[(2’,3’,4’,5’,6’- pentafluoro)fenil]porfirina e 5,15-[(2’,3’,5’,6’-tetrafluoro-4’-N-metil-p-toluenosulfamoil)fenil]-10,20- [(2’,3’,4’,5’,6’-pentafluoro)fenil]porfirina e 5,10,15-tri-[(2’,3’,5’,6’-tetrafluoro-4’-N-metil-ptoluenosulfamoil) fenil]-20-[(2’,3’,4’,5’,6’-pentafluoro)fenil]porfirina) com baixos rendimentos (0.6 a 4.5%). A título de exemplo, adotaram-se as condições otimizadas para a síntese da porfirina tetrassubstituída contendo a N-metil-p-toluenosulfonamida como substituinte, obtendo-se a correspondente 5,10,15,20-tetra-[(2’,3’,5’,6’-tetrafluoro-4’-N-metil-p-toluenosulfamoil)fenil] porfirina com um rendimento de 20%. Dado o nosso interesse no desenvolvimento de potenciais fotossensibilizadores com espectros de absorção na designada “janela terapêutica”., prosseguimos com estudos de redução de uma das porfirinas com hidrazina aquosa (NH2NH2·H2O) e cloreto de ferro(III)hexahidratado (FeCl3·6H2O), tendo-se obtido a correspondente 5,10,15,20-tetra- [2’,3’,5’,6’-tetrafluoro-4’-metanosulfamoil)fenil]clorina com rendimento de produto isolado elevado (65%). Para além disso com recurso ao método de redução de porfirinas com ptoluenosulfonilhidrazina sem solvente conseguiu-se obter a 5,10,15,20-tetra-[2’,3’,5’,6’-tetrafluoro- 4’-metanosulfamoil)fenil]bacterioclorina um rendimento de 70%. Os compostos foram caracterizados fotofísicamente, tendo revelado possuir características bastante promissoras para aplicação como fotossensibilizadores, tais como, baixos rendimentos quânticos de fluorescência (фF≤0.1375) e rendimentos quânticos de formação de oxigénio singleto adequados (фΔ≥0.59). Para avaliar a lipofilicidade e a interação com a membrana lipídica procedeuse ao cálculo do coeficiente de partição das porfirinas 5-[2’,3’,5’,6’-tetrafluoro-4’- metanosulfamoil)fenil]-10,15,20-tri-[(2’,3’,4’,5’,6’-pentafluoro)fenil]porfirina e 5,10,15,20-tetra- [2’,3’,5’,6’-tetrafluoro-4’-metanosulfamoil)fenil]porfirina através de duas metodologias, uma delas recorrendo à mistura octanol-água e a outra a vesículas unilamelares lipídicas (LUVs). Em suma, verificou-se que o número de derivatizações com grupos metanosulfonamida é crucial para modular a anfifilicidade dos compostos, sendo que estes apresentam variações significativas nos seus coeficientes de partição octanol-água (0.94≤LogP≤4). Apesar destes valores díspares, ambos apresentam uma elevada afinidade para as membranas lipídicas. Por fim, realizaram-se estudos preliminares de citotoxicidade no escuro, com a linha celular 3T3, demonstrando-se que o composto não apresenta toxicidade in vitro. No capítulo 3 desenvolve-se uma metodologia baseada no conceito de self-assembly, para a preparação de estruturas contendo porfirinas catiónicas descritas na inativação de bactérias [iodeto de 5,10,15,20-tetraquis(4-metilpiridil)porfirinato de zinco (II) e iodeto de 5,10,15,20-tetraquis(1,3- dimetilimidazol-2-il)porfirinato de zinco (II)] e a Sulfadiazina, uma sulfonamida com atividade bacteriostática comprovada. No capítulo 4 apresenta-se detalhadamente os procedimentos experimentais referentes a todos os capítulos da tese bem como a caracterização química completa de todas as moléculas sintetizadas no decorrer do trabalho (1H RMN, 19F RMN, espectrometria de massa e absorção UVVisModern society is witnessing an increasing number of drug-resistant bacteria and viruses. Due to the relevance of the subject, this work presents new methods for the synthesis of biocompatible conjugates of sulfonamides and tetrapyrrolic macrocycles for dual chemo and photodynamic therapy. The combination of these two families of compounds may occur by covalent binding or by self-assembly. Therefore, the work here presented is subdivided. The chapter 1 presents a critical and selected review of the literature underlying to differentstudy topics. The chapter 2 describes a synthetic strategy for preparing tetrapyrrolic macrocycles substituted with sulfonamides by covalent binding. First, we selected 5,10,15,20- tetrakis(pentafluorophenyl)porphyrin as starting material to carry out the methanesulfonamide derivatization optimization studies. Selective synthesis conditions were successfully obtained for the formation of 5-[2’,3’,5’,6’-tetrafluoro-4’-methanesulfamoyl)phenyl]-10,15,20-tri-[(2’,3’,4’,5’,6’- pentafluoro)phenyl]porphyrin (mono-substituted compound) or 5,10,15,20-tetra-[(2’,3’,5’,6’- tetrafluoro-4’-methanesulfamoyl)phenyl]porphyrin (tetra-substituted compound) in 19% and 70 %, yields, respectively. In order to evaluate the effect of the sulfonamide structure in the reactivity, we extended the studies using two sulfonamides previously synthesized in this work as nucleophiles: ptoluenesulfonamide and N-methyl-p-toluenesulfonamide. From this study, it was possible to synthesize and isolate the mono and disubstituted compounds of the p-toluenesulfonamide family (5-[(2’,3’,5’,6’-tetrafluoro-4’-p-toluenesulfamoyl)phenyl]-10,15,20-tri-[(2’,3’,4’,5’,6’- pentafluoro)phenyl]porphyrin, mixture of 5,10-[(2’,3’,5’,6’-tetrafluoro-4’-ptoluenesulfamoyl) phenyl]-15,20-dis-[(2’,3’,4’,5’,6’-pentafluoro)phenyl]porphyrin and 5,15- [(2’,3’,5’,6’-tetrafluoro-4’-p-toluenesulfamoyl)phenyl]-10,20-[(2’,3’,4’,5’,6’- pentafluoro)phenyl]porphyrin) and the mono-, di- and tri-substituted compounds related to substitution with the N-methyl-p-toluenesulfonamide (5-[(2’,3’,5’,6’-tetrafluoro-4’-N-methyl-ptoluenesulfamoyl) phenyl]-10,15,20-tri-[(2’,3’,4’,5’,6’-pentafluoro)phenyl]porphyrin, mixture of 5,10- [(2’,3’,5’,6’-tetrafluoro-4’-N-methyl-p-toluenesulfamoyl)phenyl]-15,20-dis-[(2’,3’,4’,5’,6’-pentafluoro) phenyl]porphyrin and 5,15-[(2’,3’,5’,6’-tetrafluoro-4’-N-methyl-p-toluenesulfamoyl)phenyl]-10,20- [(2’,3’,4’,5’,6’-pentafluoro)phenyl]porphyrin and 5,10,15-tri-[(2’,3’,5’,6’-tetrafluoro-4’-N-methyl-ptoluenesulfamoyl) phenyl]-20-[(2’,3’,4’,5’,6’-pentafluoro)phenyl]porphyrin) in low yields (0.6 to 4.5%). The optimized conditions for the synthesis of tetra-substituted porphyrin were adopted using the N-methyl-p-toluenesulfonamide as nucleophile. We obtained the corresponding 5,10,15,20-tetra[(2',3',5',6'-tetrafluoro-4'-N-methyl-p-toluenesulfamoyl) phenyl]porphyrin in 20% yield . We proceeded with the reduction studies of one of the synthesized porphyrins, using catalytic amounts of ferric chloride hexahydrate (FeCl3·6H2O) and aqueous hydrazine (NH2NH2·H2O), affording the corresponding 5,10,15,20-tetra [2 ', 3', 5 ', 6'-tetrafluoro-4'- methanesulfamoyl) phenyl] chlorin in high yield (65%). Furthermore, we used the solvent free synthetic methodology via reduction with p-toluenesulfonylhydrazide to obtain the 5,10,15,20- tetra[2 ', 3', 5 ', 6'-tetrafluoro-4'- methanesulfamoyl) phenyl]bacteriochlorin in 70% yield. The photophysical assessment shows that the compounds have very promising characteristics to be used as photosensitizers, such as low fluorescence quantum yields (фF≤0.1375) and suitable quantum yields of singlet oxygen formation (фΔ≥0.59). The lipophilicity and the interaction with the lipid membrane was evaluated by calculating the partition coefficient of 5- [2’,3’,5’,6’-tetrafluoro-4’-methanesulfamoyl)phenyl]-10,15,20-tri-[(2’,3’,4’,5’,6’- pentafluoro)phenyl]porphyrin and 5,10,15,20-tetra-[(2’,3’,5’,6’-tetrafluoro-4’- methanesulfamoyl)phenyl]porphyrin using two methods, the octanol/water partition coefficients and lipid unilamellar vesicles (LUVs). From the values obtained, we can assume that the number of metanesulfonamide fragments is crucial to modulate the amphiphilicity of the compounds. While the mono-substituted porphyrin display a logPow>4, the tetra-substituted porphyrin shows a value of log Pow =0.937. Despite these distinct values, both display high lipid membrane affinity. Finally, preliminary in vitro tests with 3T3 fibroblast cell line show that the compound is not toxic. In chapter 3 a methodology based on the self-assembly concept is developed for the preparation of structures containing cationic porphyrins described in bacteria inactivation [5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrinate zinc (II) tetraiodide, 5,10,15,20- tetrakis(1,3-dimethylimidazolium-2-yl) porphyrinate zinc (II) tetraiodide] and Sulfadiazine, a sulfonamide with proven bacteriostatic activity. The chapter 4 presents detailed experimental procedures relating to all the chapters of this thesis, as well as complete chemical characterization of all synthesized compounds (1H NMR, 19F NMR, mass spectrometry and UV-Vis absorption)

Payload holddown and release mechanism

A payload holddown and release mechanism, designated the Model 1172, was designed and built at G&H Technology during the winter of 1992/1993. The mechanism is able to restrain and release a 45-pound payload with minimal tipoff. The payload is held in place by a stainless steel band and released using electrically triggered non-explosive actuators. These actuators provide reliable operation with negligible shock and no special handling requirements. The performance of the mechanism was demonstrated in two flight tests. Data showed pitch and yaw tipoff rates of less than 0.07 radian (4 degree) per second. The Model 1172 design is an efficient replacement for conventional payload deployment devices, especially where low transmitted shock is required