Sickle Cell Hemoglobin with Mutation at {alpha}His50 Has Improved Solubility [Protein Structure and Folding]

July 16th, 2015 by Tam, M. F., Tam, T. C. S., Simplaceanu, V., Ho, N. T., Zhou, M., Ho, C.

The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial [rHb (βE6V/αH20R), and rHb (βE6V/αH20Q)], lateral [rHb (βE6V/αH50Q)] or double amino acid substitutions at both the putative axial and lateral [rHb (βE6V/αH20R/αH50Q) and rHb (βE6V/αH20Q/αH50Q)] contact sites were expressed in Escherichia coli and purified for structural and functional studies. The 1H-NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either αHis20 or αHis50 do not change the subunit interfaces or the heme pockets of the proteins. The double mutants show only slight structural alteration in the β-heme pockets. All mutants have similar cooperativity (n50), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P50) of the single mutants is comparable to that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (βE6V/αH20R) is the only mutant that has shorter delay time of polymerization and forms polymers more readily than Hb S with a Dextran-Csat value of 1.86 ± 0.20 g/dL. Hb S, rHb (βE6V/αH20Q), rHb (βE6V/αH50Q), rHb (βE6V/αH20R/αH50Q) and rHb (βE6V/αH20Q/αH50Q) have Dextran-Csat values of 2.95 ± 0.10, 3.04 ± 0.17, 11.78 ± 0.59, 7.11 ± 0.66 and 10.89 ± 0.83 g/dL, respectively. rHb (βE6V/αH20Q/αH50Q) is even more stable than Hb S under elevated temperature (60 °C).