Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant

Karin Kettisen; Maria Nyblom; Emanuel Smeds; Angela Fago; Leif Bülow

doi:10.3389/fmolb.2023.1133985

Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant

Front Mol Biosci. 2023 Mar 16:10:1133985. doi: 10.3389/fmolb.2023.1133985. eCollection 2023.

Authors

Karin Kettisen¹, Maria Nyblom², Emanuel Smeds^{3

4}, Angela Fago⁵, Leif Bülow¹

Affiliations

¹ Pure and Applied Biochemistry, Department of Chemistry, Lund University, Lund, Sweden.
² Lund Protein Production Platform, Department of Biology, Lund University, Lund, Sweden.
³ Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden.
⁴ Anesthesiology and Intensive Care, Department of Clinical Sciences, Lund University, Lund, Sweden.
⁵ Zoophysiology, Department of Biology, Aarhus University, Aarhus, Denmark.

Abstract

Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6 Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H₂O₂-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.

Keywords: DNA cleavage; X-ray crystallography; fetal hemoglobin; oxygen binding; protein surface net charge; redox reactions.

Grants and funding

This work was supported by the Swedish Research Council (VR 5607-2014, VR 2019-03996) and the Swedish Foundation for Strategic Research (RBP14-0055). The structural work was generously financed by BioCARE (a Strategic Research Area in Cancer performed at Lund University).