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Eritrocruorinas são hemoglobinas gigantes compostas por uma bicamada hexagonal de massa molecular total entre 3,0 e 4,0 MDa. A sua estrutura é baseada em unidades básicas chamadas protômeros. Doze destes compõem a partícula inteira, seis em cada camada hexagonal, resultando numa estrutura contendo 180 subunidades. Nas eritrocruorinas do tipo I, o protômero é constituído por quatro tipos de cadeias de globina: a, b, c, e d, e por três tipos de cadeias de linkers, L1, L2 e L3. A compreensão atual do seu mecanismo da ação esta atualmente prejudicada pela resolução limitada das estruturas cristalográficas disponíveis. Para abordar esta questão procuramos cristalizar uma serie de eritrocruorinas de espécies diversas visando a determinação das suas estruturas a mais alta resolução. Na primeira parte deste trabalho a eritrocruorina de Glossoscolex paulistus (HbGp) teve suas subunidades sequenciadas e a estrutura da partícula inteira resolvida a uma resolução de 3,2Å, a mais alta até o momento. Na estrutura da HbGp as quatro cadeias de globina se associam na forma de um heterotetrâmero, que se repete três vezes formando uma estrutura dodecamérica denominada cap. A estrutura do cap associada a um heterotrímero de linkers forma o então mencionado protômero. A estrutura completa permite uma descrição mais detalhada dos contatos entre subunidades que são essenciais para a manutenção da partícula como um todo. Além disto descrevemos sítios de ligação a metais (Zn2+ e Ca2+) e sítios de glicosilação, alguns dos quais são inéditos. Em seguida, a subunidade d isolada da HbGp foi cristalizada e resolvida a 2.1Å. Uma analise dos contatos cristalinos demonstra um arranjo completamente diferente daquilo visto para a subunidade d no complexo inteira. Ao invés de associar-se na formar trimeros, como acontece no complexo, a cadeia d isolada forma dímeros cristalográficos, com interface similar a aquela observada entre as cadeias d e a. Estas observações contribuíram para a compreensão de como são os possíveis mecanismos de associação das globinas para a montagem do cap. Finalmente, as eritrocruorinas de quatro outras espécies foram cristalizadas, que resultou na obtenção de uma estrutura cristalográfica preliminar para a eritrocruorina de Eisenia Andrei a uma resolução de 4.7Å. / Erythrocruorins are giant haemoglobins in the form of a hexagonal bilayer of total molecular mass between 3 and 4 MDa. Their structures are based on a basic unit called the protomer. Twelve of these comprise the entire particle, six in each hexagonal layer, resulting in a structure containing 180 subunits. In the type I erythrocruorins the protomer is composed of four types of globin chains: a, b, c e d, and three types of linker; L1, L2 e L3. Our current understanding of their mechanism of action is limited by the resolution of the crystal structures available. To address this question we have attempted to crystallize a series of erythrocruorins from different species with a view to determining a crystal structure at higher resolution. In the first part of this thesis all chains of the erythrocruorin from Glossoscolex paulistus were completely sequenced and the structure of the full particle solved at 3.2Å, the highest reported to date. In the structure the four globin chains associate to form a heterotetramer, three of which unite to form a dodecameric cap. The latter associates with a heterotrimer of linkers to form the aforementioned protomer. The full structure permits a more detailed description of the contacts between subunits which are essential for particle stability. Furthermore, we describe metal binding sites (Zn2+ e Ca2+) and glycosylation sites, some of which are have not been reported previously. Subsequently the isolated d chain was crystallized and solved to 2.1Å. An analysis of the crystal contacts shows an arrangement which is completely different to that seen in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chain associates to form a dimer across a crystallographic twofold axis making use of the interface normally used to associate with subunit a. These observations contributed to our understanding of the possible mechanisms of association of globin chains during the formation of the cap. Finally, the erythrocruorins from four other species were crystallized, which resulted in the preliminary determination of the structure of that from Eisenia Andrei at 4.7Å.

New Zealand's five endemic mudfish (Neochanna spp.) species have distributions that differ both geographically and by habitat type. Differences in habitat preferences between species have led to the proposal of an evolutionary series within the group. A morphological cline can be observed from the galaxiform Chatham Island and Canterbury species inhabiting lakes and streams, respectively, to the anguilliform Northland and brown mudfishes of ephemeral wetlands. Morphological specializations proposed for wetland dwelling include loss of pelvic fins, reduced eyes, enlarged nostrils, development of caudal flanges, and elongation of dorsal and anal fin bases to become almost confluent with the caudal fin. Another expectation of adaptation to wetland dwelling is specializations in respiratory physiology to obtain oxygen from highly hypoxic or acidic waters, and the ability to cope with seasonal exposure to air during the drought season. Expected respiratory specializations to wetland dwelling include high oxygen affinity haemoglobins, high levels of cooperative oxygen binding, the presence of multiple haemoglobins and the ability to aestivate and survive long periods of emersion. The four mainland Neochanna species were examined to determine if differences in haemoglobin expression as well as differences in haemoglobin oxygen binding correlated with differing habitats and treatments. Whole blood oxygen affinity was determined at several pH levels (6.5, 7.0, 7.5 and 8.0) and temperatures (10'C, 15'C and 20'C), as well as different treatments (aestivating, fasting and control) using a Hemox analyzer. The presence of multiple haemoglobins was determined by isoelectric focusing. All four species displayed high oxygen affinities (p50 = 6.5 to 9.5 mm Hg at pH 7.5 15'C), moderate levels of cooperativity (Hill coefficients = 1.75 to 2.00 at pH 7.5 15'C), pH sensitivity (Bohr coefficients = -0.62 to -0.94 between pH 7.5 and 7.0 at 15'C), temperature sensitivity (ΔH = -2.20 to -15.78 k cal mol-1 between 10'C and 15'C) and the presence of multiple haemoglobins. Black, brown and Northland mudfish were able to survive aestivation for six weeks but there were no changes between air-breathing and water-breathing individuals with respect to oxygen binding characteristics. Although there is evidence of habitat specialization in haemoglobin physiology between mudfish species, differences between species did not correlate with the evolutionary series proposed for specialization to dwelling in ephemeral wetlands and latitudinal distributions of mudfish species appear to strongly dictate oxygen binding properties of mudfish whole blood.

Eritrocruorinas são hemoglobinas gigantes compostas por uma bicamada hexagonal de massa molecular total entre 3,0 e 4,0 MDa. A sua estrutura é baseada em unidades básicas chamadas protômeros. Doze destes compõem a partícula inteira, seis em cada camada hexagonal, resultando numa estrutura contendo 180 subunidades. Nas eritrocruorinas do tipo I, o protômero é constituído por quatro tipos de cadeias de globina: a, b, c, e d, e por três tipos de cadeias de linkers, L1, L2 e L3. A compreensão atual do seu mecanismo da ação esta atualmente prejudicada pela resolução limitada das estruturas cristalográficas disponíveis. Para abordar esta questão procuramos cristalizar uma serie de eritrocruorinas de espécies diversas visando a determinação das suas estruturas a mais alta resolução. Na primeira parte deste trabalho a eritrocruorina de Glossoscolex paulistus (HbGp) teve suas subunidades sequenciadas e a estrutura da partícula inteira resolvida a uma resolução de 3,2Å, a mais alta até o momento. Na estrutura da HbGp as quatro cadeias de globina se associam na forma de um heterotetrâmero, que se repete três vezes formando uma estrutura dodecamérica denominada cap. A estrutura do cap associada a um heterotrímero de linkers forma o então mencionado protômero. A estrutura completa permite uma descrição mais detalhada dos contatos entre subunidades que são essenciais para a manutenção da partícula como um todo. Além disto descrevemos sítios de ligação a metais (Zn2+ e Ca2+) e sítios de glicosilação, alguns dos quais são inéditos. Em seguida, a subunidade d isolada da HbGp foi cristalizada e resolvida a 2.1Å. Uma analise dos contatos cristalinos demonstra um arranjo completamente diferente daquilo visto para a subunidade d no complexo inteira. Ao invés de associar-se na formar trimeros, como acontece no complexo, a cadeia d isolada forma dímeros cristalográficos, com interface similar a aquela observada entre as cadeias d e a. Estas observações contribuíram para a compreensão de como são os possíveis mecanismos de associação das globinas para a montagem do cap. Finalmente, as eritrocruorinas de quatro outras espécies foram cristalizadas, que resultou na obtenção de uma estrutura cristalográfica preliminar para a eritrocruorina de Eisenia Andrei a uma resolução de 4.7Å. / Erythrocruorins are giant haemoglobins in the form of a hexagonal bilayer of total molecular mass between 3 and 4 MDa. Their structures are based on a basic unit called the protomer. Twelve of these comprise the entire particle, six in each hexagonal layer, resulting in a structure containing 180 subunits. In the type I erythrocruorins the protomer is composed of four types of globin chains: a, b, c e d, and three types of linker; L1, L2 e L3. Our current understanding of their mechanism of action is limited by the resolution of the crystal structures available. To address this question we have attempted to crystallize a series of erythrocruorins from different species with a view to determining a crystal structure at higher resolution. In the first part of this thesis all chains of the erythrocruorin from Glossoscolex paulistus were completely sequenced and the structure of the full particle solved at 3.2Å, the highest reported to date. In the structure the four globin chains associate to form a heterotetramer, three of which unite to form a dodecameric cap. The latter associates with a heterotrimer of linkers to form the aforementioned protomer. The full structure permits a more detailed description of the contacts between subunits which are essential for particle stability. Furthermore, we describe metal binding sites (Zn2+ e Ca2+) and glycosylation sites, some of which are have not been reported previously. Subsequently the isolated d chain was crystallized and solved to 2.1Å. An analysis of the crystal contacts shows an arrangement which is completely different to that seen in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chain associates to form a dimer across a crystallographic twofold axis making use of the interface normally used to associate with subunit a. These observations contributed to our understanding of the possible mechanisms of association of globin chains during the formation of the cap. Finally, the erythrocruorins from four other species were crystallized, which resulted in the preliminary determination of the structure of that from Eisenia Andrei at 4.7Å.