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Date Permissions Signed


Date of Award


Document Type

Masters Thesis

Degree Name

Master of Science (MS)



First Advisor

Anthony-Cahill, Spencer J.

Second Advisor

Spiegel, P. Clint

Third Advisor

Smirnov, Sergey L.


The tetrameric composition of human hemoglobin complicates protein engineering efforts that are required to improve its potential as an oxygen-carrying therapeutic. In our research to design a single-chain version of the hemoglobin molecule (scHb), we have co-expressed a circularly permuted human β-globin (cp-β) with human α-globin. At micromolar concentrations, the purified recombinant globins appear to associate to form an α-cpβ heterodimer in solution rather than the expected α2-cpβ2 heterotetramer. Compared to recombinant human hemoglobin, the α-cpβ heterodimer exhibits a stronger ligand binding affinity. Knowledge of the intermolecular interactions favoring formation of the α-cpβ heterodimer will be instrumental in understanding the global structural consequences of the cpβ mutation, and in directing future protein engineering efforts to optimize the function of permuted hemoglobins. X-ray diffraction of α-cpβ crystals has been employed to determine the molecular structure of this protein complex at near-atomic resolution (2.7 Å). Examination of the structure shows that, in the crystal, the subunits associate to form a heterotetramer similar to that of wild-type hemoglobin in the high affinity "R-state". The structure also confirms the incorporation of the β-globin His146 (wild-type numbering) into the cpβ linker, which removes an important ionic interaction that stabilizes the low oxygen affinity (T state) conformation. Structural information obtained as a result of this work will guide future protein engineering efforts to enhance cooperativity of oxygen binding and the T-state stability of future hemoglobin constructs.





Western Washington University

OCLC Number


Subject – LCSH

Hemoglobin--Reactivity; Hemoglobin; Oxygen--Physiological transport; Protein binding; Iron proteins; Ligand binding (Biochemistry)




masters theses




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