The objective of this research is to further investigate the mechanisms of polymerization of deoxy-Hb S using biochemical, biophysical and recombinant DNA technologies. We will also attempt to define whether human biochemical, biophysical and recombinant DNA technologies. We will also attempt to define whether human embryonic Hb (Gower-Hb 2, alpha2 epsilon2) can inhibit Hb S polymerization. The long-range goals of this research are to understand the mechanism of polymerization of HbS and to generate the basic knowledge that will aid in the development of anti-sickling agents in the future. In the previous funding period our laboratory has made considerable progress in further understanding the mechanism of polymerization on the molecular level using biochemical, biophysical and recombinant DNA technologies. We have succeeded in characterizing various recombinant Hbs with substitutions at the beta6, beta7, beta85, beta87 and beta88 positions in Hb S as well as gamma6 and gamma87 in Hb F and delta6 and delta87 of Hb A/2 using a yeast expression system. We have also succeeded in expressing single soluble human beta chains in bacteria. These systems coupled with PCR-based mutagenesis will facilitate production of sufficient amounts of hemoglobin for the studies proposed in this grant.
Specific Aims of this application are as follows: (1) To further understand the strong hydrophobic interaction of beta6 Val in Hb S polymers we will further clarify the critical role of Phe beta85 and Leu beta88 in the acceptor pocket. We will define the structure of the EF acceptor pocket of various recombinant Hb S variants containing substitutions at beta85 and beta88 positions in yeast by X-ray analysis of crystals. We will study the effect of amino acid replacement at the beta85 and beta88 positions on polymer structure using electron microscopy. We will further study the effects of the various amino acid replacements at the acceptor and donor sites for hydrophobic interactions on polymerization and polymer structure under various experimental conditions in order to further clarify the critical role of Phe beta85 and Leu beta88 in the acceptor pocket. (2) We will establish methods to demonstrate the presence of nuclei prior to polymerization using a variety of physical methods. We will also characterize formation of nuclei prior to HbS polymerization. The relationship between protein-protein interactions and nuclei formation will also be studied. (3) We will also evaluate anti- sickling properties of epsilon-globin of embryonic Hb compared to the gamma-globin chain of Hb F, based on the presence of a highly polar Lys at epsilon87 instead of beta87 Thr or gamma87 Gln. We will express human embryonic hemoglobin (alpha2 epsilon2) using a yeast expression system. Our rationale for testing epsilon-globin anti-sickling properties is that transcriptional control of this gene is autonomous in transgenic mice; and there expression of this gene in adult life may be much easier to realize compared to the fetal globin genes. These studies will further provide the basic knowledge of the molecular pathology of sickle cell disease and possibility to generate the basis to formulate strategies aimed at inhibition of polymerization of deoxy Hb S in future.
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