The overall goals of this project are (a) the identification and characterization of those intermolecular contact sites that are energetically important in the deoxy sickle hemoglobin (HbS) aggregation process through direct detection of Hb motions and motional restrictions within the polymer state, (b) the use of non-covalent HbS gelation inhibitors as stereochemical probes for energetically important HbS polymerization sites through a determination of binding sites and stereochemistry, under solution conditions, for such inhibitors, with particular emphasis on a nitroxide spin labeled analog of phenylalanine (SL-Phe) that we have developed, (c) to further characterize the molecular stereochemistry of the nucleation process that appears to initiate HbS aggregation, and (d) to refine the application of saturation transfer EPR and pulsed EPR methods to provide more quantitative descriptions of slow macromolecular motions, both for deoxy HbS, and for general applications. Conventional, saturation transfer, and pulsed EPR (electron spin echo and pulse ELDOR) with 15N,2H-substituted spin labels, and multinuclear NMR spectroscopies will be used. The long range goals are to suggest routes for developing improved therapeutic procedures in sickle cell anemia through a more detailed molecular characterization of sickle hemoglobin aggregation mechanisms, and to develop improved spin label EPR methods for studying macromolecular motions by refining saturation transfer EPR methodology through a variety of methods to enhance resolution and interpretation, and by developing the use of pulsed EPR methods in relatively well-defined biomolecular systems.
