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Polymerization of deoxy-sickle cell hemoglobin in high-phosphate buffer.
1Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Journal of Structural Biology
|October 29, 2000
Summary
Deoxy-sicklecell hemoglobin (HbS) polymerization forms similar helical fibers in both high and low phosphate buffers. High phosphate conditions allow polymerization studies with less HbS, aiding mutant analysis.
Area of Science:
- Biochemistry
- Structural Biology
- Hematology
Background:
- Deoxy-sicklecell hemoglobin (HbS) polymerization is central to sickle cell disease pathogenesis.
- Understanding HbS polymer structure is crucial for developing therapeutic strategies.
- Previous studies focused on polymerization under physiological conditions (0.05 M phosphate).
Purpose of the Study:
- To characterize the structure of HbS polymers formed in 1.5 M phosphate buffer.
- To compare HbS polymer structures formed under high and low phosphate conditions.
- To assess the utility of high phosphate conditions for studying HbS polymerization, especially with limited mutant samples.
Main Methods:
- Site-directed mutagenesis of HbS.
- Induction of HbS polymerization in 0.05 M and 1.5 M phosphate buffers.
- Cryo-electron microscopy (cryo-EM) of frozen-hydrated HbS polymers.
- Negative staining electron microscopy.
- Fourier transform analysis of micrographs.
- 3D reconstruction of HbS fibers.
Main Results:
- HbS fibers are the primary polymerization product under both high (1.5 M) and low (0.05 M) phosphate conditions.
- Fourier transforms and cryo-EM reconstructions show similar periodicities and 14-strand structures for fibers formed in both conditions.
- High phosphate conditions enable polymerization at significantly lower HbS concentrations (ca. 5 mg/ml vs. 165 mg/ml).
- A minor 10-strand fiber variant was observed in high phosphate conditions.
- HbS polymer crystals formed under both conditions share identical unit cell parameters.
Conclusions:
- HbS polymerization in 1.5 M phosphate buffer yields structures largely indistinguishable from those formed in 0.05 M phosphate buffer.
- High phosphate conditions offer a valuable, low-concentration method for studying HbS polymerization and its structural intermediates.
- Cryo-EM of frozen-hydrated specimens is essential for accurate structural determination, overcoming limitations of negative staining.