Structure and function of hemoproteins from cold-adapted organism
Russo, Roberta (2011) Structure and function of hemoproteins from cold-adapted organism. [Tesi di dottorato] (Inedito)
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Environmental oxygen availability certainly plays a key role in the evolution of polar marine life, as suggested by the physiological and biochemical strategies that the organisms have adopted to acquire, deliver and scavenge oxygen. The psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 gives the opportunity to explore the cellular strategies adopted in vivo by cold-adapted microorganisms to cope with cold and high oxygen concentration. Within vertebrates, the dominant suborder Notothenioidei of the Southern Ocean is one of the most interesting models to study the evolutionary biological responses to extreme environment. Hemoproteins of cold-adapted organisms are likely to fulfil important physiological roles, not only in delivering oxygen to cells, but also in protecting them from the nitrosative and oxidative stress. This thesis will in particular focus on: (i) the structural and functional features of globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125, (ii) the role of neuroglobin (Ngb) recently identified in the brain of Antarctic notothenioid fish. The genome of the cold-adapted bacterium P. haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins (Hbs) exhibiting a 2/2 -helical fold (2/2Hb). One of these 2/2Hb (Ph-2/2HbO) has been over-expressed and characterised by spectroscopic analysis, kinetic measurements and computer simulation approaches (Howes et al., 2011; Giordano et al., 2011). The results indicate unique adaptive structural properties, that overall confer higher flexibility to the protein and may facilitate its functioning in the cold by providing greater freedom for the correct positioning of ligand(s). Similar to Ngb, the recombinant protein is hexacoordinated in the ferric and ferrous forms, and shows a strong dependence on pH (Howes et al., 2011; Giordano et al., 2011). Polar fish are a suitable model to learn more about the function of globins in the brain, and especially about their role in species devoid of Hb and Myoglobin (Mb). The finding that Antarctic icefishes retain the Ngb gene despite having lost Hb, and Mb in most species, suggests a crucial function. The function of Ngb needs to be ascertained, because it may have important implications in the physiology and pathology of the brain. The first structural model of fish Ngb was described using molecular dynamics simulations. Specifically, Ngb genes from a colourless-blooded Antarctic icefish species (Chaenocephalus aceratus), and a related red-blooded species (Dissostichus mawsoni), were cloned, the recombinant proteins were expressed and purified, and then sequenced and analysed. Both Antarctic fish Ngbs are hexacoordinated, but have some peculiarities that differentiate them from mammalian counterparts: they have extensions in the N and C termini, interacting with the EF loop, and a gap in the alignment that changes the CD-region structure/dynamics, that has been found to play a key role in human Ngb. The adaptive modifications to compensate for the effects of low temperature appear to primarily rely on a higher flexibility of key parts of the molecular structure and/or decreased overall stability. At all levels analysed, the functionally most crucial adaptation to permanently low temperatures apparently requires molecular flexibility to support cell functioning. Proteins are the major targets for the ensuing mechanisms of adaptation.
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