GSBS Dissertations and Theses

Title

Crystallographic Studies of the Pathological Polymerization of Human Hemoglobin

Approval Date

2-1-1998

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Department of Biochemistry and Molecular Biology

Subjects

Hemoglobins; Crystallography; Academic Dissertations

Abstract

Sickle cell disease is caused by the intracellular polymerization of human hemoglobin containing a mutation of a glutamic acid to a valine residue at the sixth position of the β chain. This substitution of a hydrophobic residue for a hydrophilic residue greatly decreases the solubility of the hemoglobin tetramer, promoting the formation of ordered fibers in deoxygenated red blood cells. These fibers are composed of double strands of hemoglobin tetramers, which effectively bury the valine side chain in a hydrophobic pocket, eliminating its unfavorable interaction with solvent. Inhibition of fiber formation would greatly alleviate the symptoms of sickle cell disease; therefore, the elucidation of the structure of the fiber is critical to developing treatments for the disease. In this thesis, an analysis of the crystal structures of the double strand component of the fibers formed by HbS and other site-directed mutants displaying altered sickling properties is undertaken.

Structure of HbS at high resolution: The structure of HbS was solved at 2.05 Å resolution. This high-resolution analysis produced significant improvements in the previous 3.0 Å model. In particular, the accurate positioning of side chains, and the placement of more than 500 solvent molecules was achieved. Some side chains in the physiologically relevant contacts were moved by as much as 3.5 Å away from their locations in the lower resolution model. The structure also demonstrates that well ordered water molecules are located in both the axial and lateral contacts, some of which may be exploited in the design of sickling inhibitors.

Structures of Hbβ6L and Hbβ6W: The crystal structures of two human hemoglobins with mutations at the β6 position that display altered sickling behavior were solved and refined at high resolution. Both mutants were crystallized under conditions similar to those used with HbS. Hbβ6L showed a greater tendency to polymerize, and displayed reduced delay times in aggregation assays. The refined structure was very similar to that of HbS, with changes confined mostly to the lateral contacts. However, the packing of Leu β6 in the linear crystalline double strands is sub-optimal. This has important ramifications for the differences between the crystalline double strands and those within the physiological HbS fiber. Hbβ6W, on the other hand, showed a reduced tendency to polymerize and shorter delay times in polymerization assays. The protein crystallized in a different space group from the other two mutants (HbS and Hbβ6L) under the same crystallization conditions, and formed fundamentally different double strands in the crystals. This structure demonstrates conserved interactions at the axial contacts, but very different interactions at the lateral contacts. The alternate double strand formed by this protein in the crystal may be useful as a model for tetramer interactions within the physiological fiber, or as a structural model for heterogeneous nucleation. The structures of each of these mutants help explain their altered polymerization behavior.

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