HOW SITE DIRECTED MUTANTS AFFECT HBS FIBER STRUCTURE

Yeshiva University, New York NY

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Abstract

DESCRIPTION (provided by applicant): 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.