Previous work by the applicant has suggested that a single model consisting of a hierarchy of globin complexes can adequately describe the quaternary structure of three representative, ca.3,6OOkDa, hexagonal bilayer (HBL) molecules: the hemoglobins (Hbs) of the earthworm Lumbricus terrestris and the leech Macrobdella decora and the chlorocruorin (Chl) of the marine polychaete Eudistylia vancouverii. In this model, 12 ca.2OOkDa complexes, each a dodecamer of approximately 16kDa, heme-- containing chains (144 globin chains and 144 heme groups) are linked together by 30 to 40 chimeric globin chains (ca.3OkDa) and 30 to 40 Ca(II), into an HBL structure. The dodecamers in turn consist of smaller subunits: 3 monomers and 3 disulfide-bonded trimers in Lumbricus Hb, 6 monomers and 3 disulfide-bonded dimers in Macrobdella Hb or 3 disulfide-- bonded tetramers in Eudistylia Chl; although the dodecameric complexes exhibit substantial cooperativity of oxygen binding, full cooperativity is dependent on the presence of a complete HBL structure. The objectives of the proposed research can be grouped as follows. (A) The description of the pathways of the dissociation and reassociation of HBL structures, including the effect of group IIA cations and the determination of the number of Ca-binding sites and their affinities in the HBL's and their subunits. (B) The determination of the crystal structures of the dodecamer and/or smaller subunits of the HBL's by x-ray diffraction, accompanied by modeling of the small subunits of Lumbricus Hb based on known globin crystal structures. (C) The determination of the kinetics of binding of 02, CO and NO to the HBL's and their subunits, measurement Of 02 binding of the reassociated HBL structures and subunits and examination of alterations in the molecular shape of HBL's and their subunits attendant upon ligand addition or removal.
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