HBS FIBER QUINTIC STRUCTURE--BLUEPRINT FOR THERAPEUTIC INTERVENTION

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The mutation of Glu-6(beta) of HbA to Val endows the mutated A helix a new 'quinary structural memory'. The translation of this information to generate the HbS fiber occurs only in the deoxy conformation of the protein. Both crystallographic and electron microscopic studies have identified Wishner-Love double strand as the 'structural motif' of deoxy HbS fiber. HbS fiber models implicate Intra-double strand contacts as predominantly betaS chain contacts whereas the inter double strand contacts as predominantly alpha-chain contacts. There is a higher degree of correlation of the implicated intra-double strand contact regions with the results of the copolymerization studies of HbS, whereas for the inter double strand contacts, the correlation is very poor. Attempts will be made to resolve the discrepancy between the solubility studies and the implicated contact regions of HbS fiber of the contact sites alpha6, alpha11, and alpha23. The propensity of various amino acid side chains introduced at this site to inhibit polymerization will be established. The "photoaffinity functional labelling" of the implicated contact region with their complementary "surfaces in the fiber" using site specifically introduced affinity labels at these sites will be pursued. We also propose to introduce multipoint mutations into HbS by exchanging human alpha chains with other mammalian alpha-chains. The polymerization inhibiting potential of mouse alpha-chain has been well recognized. Using segment condensation approach and employing mouse and horse alpha- chain as models, we will delineate the influence of mutations of the alpha-chain, particularly those at the junction of A and B helix and EF corner, as the dominant contributors for the inhibiting potential of the mouse and/or horse alpha-chains. Electrostatic modification of the amino terminal domain of the beta-chain of HbS by acetylation and succinylation of Val-1(beta), as well as ethylation and carboxy methylation will be carried out and the influence of respective modifications on solubility and kinetics of polymerization will be investigated to unravel the possible role of changes in the macro-dipole moment at the amino termines of A-helix in the 'translation of the quinary structural memory' of the mutated segment. 19F labelled trifluoroacetyl and trifluoroethyl groups will be introduced on Val-1(beta) of HbA and HbS to monitor the possible differences in the microenvironment of the two proteins at their amino terminus in the oxy and deoxy conformation. The studies discussed here will increase our understanding of the quintic structure of HbS and the microenvironment of the crucial side chains of contact regions, the modification of which are likely to have the maximum influence on the propensity of HbS to polymerize.