The intracellular polymerization of sickle cell hemoglobin to form rod-like fibers causes sickle cell anemia. Cells containing fibers become distorted and rigid and occlude small capillaries causing tissue destruction. The structure of the fibers is of interest since this information could provide a rational basis for therapies aimed at inhibiting fiber formation. In addition, understanding the structural basis of HbS polymerization offers insights relevant to other systems involving macromolecular assemblies. We have succeeded in carrying out reconstructions of electron micrographs of frozen-hydrated HbS fibers. By averaging 10 fibers into the reconstruction we have improved the resolution of the reconstruction from 32A (previously published) to 16A radially and 8A axially. The improvement in resolution is caused by increasing the signal to noise ratio. We believe that the frozen-hydrated fibers are sufficiently well preserved that we could attain a resolution of at least 4 -6A by averaging several hundred particles into the reconstruction. A second series of experiments involves synthesis of double mutants (the HbS site and one other site) to explore how changes in individual amino acids effect the fiber properties. Site directed mutants at intermolecular contacts provide a means of critically testing current molecular models of the fiber structure by their effect on polymerization. We propose to study the structure of fibers containing the site directed mutants by electron microscopy and polymerization kinetics by video enhanced light microscopy.
| 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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