This proposal describes the use of recombinant DNA (for single-site substitutions or for regional substitutions of larger segments and novel chemical modifications to study sickle hemoglobin (HbS) polymerization. The important regions of HbF, of current clinical interest and of HbA2, in inhibition of HbS polymerization and their relative strengths will be evaluated. Our recent finding of the 70-fold lower tetramer-dimer dissociation constant of HbF compared to HbS, HbA and HbA2 will be expanded. Studies will be continued on a hybrid HbA/F containing the amino acids of the a1gamma1 and alpha1gamma2 interfaces of HbF and the remaining sequence of the beta-chain. The mechanism by which the subunit interface properties are influenced by other parts of the sequence will be studied. A candidate is helix A (N-terminal amino acids 1-18) of the chain whose contribution both to subunit interface properties and to HbS polymerization will be evaluated. The a source of the proton uptake for both HbA and HbF dissociating to dimers will be investigated by mutagenesis of potential sites common to both Hbs. Dimer to monomer dissociation will continue to be studied with the aid of a recombinant Hb with a substitution in the alpa1beta1 interface. Inhibition of polymerization with recombinant HbS mutants will focus on obtaining quantitative information on the contributions of sites known to be at contact points between tetramers, either inter or intra-strand. We propose to establish which sites function independently and whether they are at lateral or axial contacts in order to identify them as important targets for anti-sickling agents. The second region that we propose to study is the central cavity where we will identify the most important sites in keeping HbS in the more open (sickling) conformation. A novel class of chemical anti-polymerization inhibitors directed at the central cavity will be synthesized based on familiar chemistry and tested to maintain the central cavity of HbS less open (non-sickling). Certain recombinant mutants with either selective or regional substitutions will be supplied to our collaborators for crystallographic studies (Rover), electron microscopy (Josephs) or kinetics of polymerization (Ferrone).

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL018819-27A1
Application #
2764031
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1982-07-01
Project End
2002-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
27
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Northeastern University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02115
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
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
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
Manning, Lois R; Russell, J Eric; Padovan, Julio C et al. (2007) Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell. Protein Sci 16:1641-58
Ashiuchi, Makoto; Yagami, Takeshi; Willey, Ronald J et al. (2005) N-terminal acetylation and protonation of individual hemoglobin subunits: position-dependent effects on tetramer strength and cooperativity. Protein Sci 14:1458-71
Geva, Alon; Clark, Jennifer J; Zhang, Yuxun et al. (2004) Hemoglobin Jamaica plain--a sickling hemoglobin with reduced oxygen affinity. N Engl J Med 351:1532-8
Zhang, Yuxun; Manning, Lois R; Falcone, Jill et al. (2003) Human erythrocyte membrane band 3 protein influences hemoglobin cooperativity. Possible effect on oxygen transport. J Biol Chem 278:39565-71
Manning, L R; Manning, J M (2001) The acetylation state of human fetal hemoglobin modulates the strength of its subunit interactions: long-range effects and implications for histone interactions in the nucleosome. Biochemistry 40:1635-9
Chen, W; Dumoulin, A; Li, X et al. (2000) Transposing sequences between fetal and adult hemoglobins indicates which subunits and regulatory molecule interfaces are functionally related. Biochemistry 39:3774-81
Li, X; Himanen, J P; Martin de Llano, J J et al. (1999) Mutational analysis of sickle haemoglobin (Hb) gelation. Biotechnol Appl Biochem 29 ( Pt 2):165-84

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