The overall goal of this project is to characterize the structure and assembly mechanisms of the deoxy HbS polymer and gel in structure in within the red cells.
Specific aims are: 1) Exploring the properties and functional consequences of the polymer water compartment (PWC). The idea is based on model studies predicting a volume shift associated with polymerization. Using C-70kD dextran the concentration of deoxy-HbS in the polymer, Cp, was calculated to be 54.7 g/dl. This value is lower than the concentration of Hb in the red cells (136 g/dl), and implies the presence of polymer associated water. It is proposed that this is associated with compartmentalization of red cell components. In particular, Dr. Bookchin wants to investigate the redistribution of organic phosphates, in view of their reported effect on enhancing gelation, and to assess whether changes in metabolic activity are associated with gelation of the deoxy HbS in the red cells. 2) Testing the variability of Cp in deoxy HbS alone and in mixture with other hemoglobins either chemically modified or variants produced by recombinant techniques. The idea is based on preliminary data in which 1:1 mixtures of HbS and HbA or HbC or HbF show an increase in Csat. 3) Implementing the Csat determination in the presence of dextran to microquantities and correlate it with the values obtained from the micro- kinetic assay of Dr. Ferrone. 4) Testing the role of membrane associated sites in the heterogenous nucleation of HbS. For these studies three types of RBC membranes will be prepared, inside out (IOVs) and right side out ghosts in which all the membrane components are preserved, cytoskeleton- free IOVs from which the cytoskeletal proteins have been released, and hybrid erythrocytes deoxygenation. 5) The identification of intra and inter contact points in the fibers, will be done focussing on sites which are susceptible to chemical modifications. A large variety of chemical modifications, either specific or introduced through a semisynthetic approach are proposed as a) amidation of specific Glu residue, b) construction of chimeric hemoglobins c) photoaffinity labeling of contact regions d) preparation of crosslinked asymmetric hybrids.
| Yosmanovich, Donna; Rotter, Maria; Aprelev, Alexey et al. (2016) Calibrating Sickle Cell Disease. J Mol Biol 428:1506-14 |
| Tokarev, Alexander; Aprelev, Alexey; Zakharov, Mikhail N et al. (2012) Multifunctional magnetic rotator for micro and nanorheological studies. Rev Sci Instrum 83:065110 |
| Manning, James M; Popowicz, Anthony M; Padovan, Julio C et al. (2012) Intrinsic regulation of hemoglobin expression by variable subunit interface strengths. FEBS J 279:361-9 |
| Weng, Weijun; Ferrone, Frank A (2011) Metastable gels: A novel application of Ogston theory to sickle hemoglobin polymers. Biophys Chem 154:99-101 |
| Rotter, Maria; Yosmanovich, Donna; Briehl, Robin W et al. (2011) Nucleation of sickle hemoglobin mixed with hemoglobin A: experimental and theoretical studies of hybrid-forming mixtures. Biophys J 101:2790-7 |
| Rotter, Maria A; Chu, Haiyan; Low, Philip S et al. (2010) Band 3 catalyzes sickle hemoglobin polymerization. Biophys Chem 146:55-9 |
| Zakharov, Mikhail N; Aprelev, Alexey; Turner, Matthew S et al. (2010) The microrheology of sickle hemoglobin gels. Biophys J 99:1149-56 |
| Manning, Lois R; Popowicz, Anthony M; Padovan, Julio et al. (2010) Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces. Protein Sci 19:1595-9 |
| Wang, Jiang Cheng; Kwong, Suzanna; Ferrone, Frank A et al. (2009) Fiber depolymerization: fracture, fragments, vanishing times, and stochastics in sickle hemoglobin. Biophys J 96:655-70 |
| Manning, Lois R; Russell, J Eric; Popowicz, Anthony M et al. (2009) Energetic differences at the subunit interfaces of normal human hemoglobins correlate with their developmental profile. Biochemistry 48:7568-74 |
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