Structural Studies to Understand the Effects of N108K T-State Stabilizing Mutation in Circularly Permuted Hemoglobin

The primary goal of our project is to develop a functional hemoglobin based oxygen carrier (HBOC) for clinical care. In order to accomplish this, we are working on designing a single chain hemoglobin (scHb) that can serve as the building block of a monodisperse polymeric HBOC. The scHb is formed from novel covalent fusions between the two α chains and two circularly permuted β chains. The initial constructs of non-covalently linked α and cp-β showed decreased stability and low T-state affinity (Asmundson, et. al., 2009). To counter these effects, two point mutations have been included to increase T-state affinity (Tsai et. al in 1999). The first mutation, N108K, shows greater T-state affinity, however the second mutation, V96W, shows decreased T-state affinity. I am conducting X-ray crystallography trials of the βN108K mutant in order to understand the structural basis for this unexpected effect on the T-state stability, and to inform future protein engineering of the HBOC candidates.