Structure and function of tetrameric hemoglobins and their mutants at a molecular and cellular level.
Balsamo, Anna (2011) Structure and function of tetrameric hemoglobins and their mutants at a molecular and cellular level. [Tesi di dottorato] (Inedito)
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The present Ph.D. thesis has focused on tetrameric hemoglobins (Hbs), both recombinant and natural, both from human origin and Antarctic fish, using a multidisciplinary approach based on spectroscopic, crystallographic and computational techniques. In particular the main scope of the research has been the elucidation of two still unsolved problems in the chemistry of tetrameric Hbs: 1) the role of the bis-histidyl heme coordination in the Hb function and oxidation process and 2) the role of the tertiary and quaternary structure in the modulation of the Root effect (namely drop of oxygen affinity with loss of cooperativity at low physiological pH). The first topic has been mainly approached through a comparative experimental (spectroscopic and crystallographic) / computational study of the β-subunits of human hemoglobin (β-HbA) and of the recombinant β-subunits Hb from Antarctic fish Trematomus bernacchii (β-HbTb), whose heterotetramer, in the ferric state, forms a mixture of aquo-met at the α-subunits and bis-histidyl adduct at the β-subunits. Similarly to the human β-chains, β-HbTb self-assembles to form the homotetramer (β4-HbTb); however, the latter quantitatively forms reversible ferric and ferrous bis-histidyl adducts, which are only partially present in the human tetramer (β4-HbA). The molecular dynamics study of the isolated β-subunit of the two Hbs indicates that the ability to form hemichrome is an intrinsic feature of the chain; moreover, the greater propensity of β-HbTb to form the bis-histidyl adduct is probably linked to the higher flexibility of the CD loop region. These findings are in perfect agreement with the X-ray structure of β4-HbA in the ferric (solved in this thesis) that hosts only aquo-met coordination. The mechanism of hemichrome formation was also investigated by determining the x-ray crystal structure of the fully oxidized Hb from Antarctic fish Trematomus newnesi and by performing a Resonance Raman microscopy study on the Hb crystals from a sub-Antarctic fish (Eleginops maclovinus). The second topic has been investigated through a combined spectroscopic (resonance Raman (RR) /Vis microspectroscopy) and X-ray crystallographic study of oxygen (O2) and nitric oxide (NO) binding to the T-state HbTb (Root-effect Hb) crystals at two different pHs, 6.0 and 8.4. The choice of these pH values is justified by the known tertiary structural differences of their X-ray structure in deoxygenated forms at different pH values. Oxygen binding curves have shown that the low -to- high pH transition induces 2-3 fold increase in oxygen affinity and that about half of the cooperativity present in solution is retained in the crystal. The simultaneous X-ray data collection, on the T-state nitrosyl-HbTb crystals assisted by online Raman acquisition, at the synchrotron Swiss Light Source (PXII beamline), clearly indicates an X-ray induced NO-photodissociation, providing a general physical explanation of the low content of nitrosyl-hemoglobin structures deposited in the Protein Data Bank. RR microscopy supported crystallography in choosing the X-ray dose feasible to collect a fully nitrosylated hemoglobin. The comparison between fully NO ligated and photolized crystal refined structures provides an elegant evidence of heme-heme communication mediated via the CDα region, that involves those interactions that were suggested to be contributor to the Root effect in HbTb. In addition this Ph.D. thesis also focused on a study at cellular level (via Raman imaging and Optical Tweezers) of healthy and C / S anemic erythrocytes [containing HbS (β6Glu → Val) and HbC (β6Glu → Lys)]. The different photo-sensibility and rigidity of healthy, C and S erythrocytes showed that the Raman micro-spectroscopy analysis combined with the use of Optical Tweezers offers enormous potential in the diagnosis of normal and diseased cells.
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