The long-term goal of this research is the design and synthesis of a single-chain recombinant human hemoglobin (sc-rHb) which binds oxygen reversibly and cooperatively. The sc-Hb design is based on insertion of a circularly permuted ?-globin ("cp?") into a surface loop of human ?- globin to form an ?-cp? dimer. Compared to normal hemoglobin, the proposed sc-rHb could be more easily engineered for optimum performance as a red cell substitute. The development of a sc-rHb molecule would be a significant achievement in protein engineering as well as a step forward in the development of oxygen delivery therapeutics that would be of significant value in critical care. The specific aims of the proposed research are: (1) Optimize expression yields for mutant globins using a combination of altered growth conditions and site-directed mutations shown to improve expression yields for recombinant Hb;(2) Optimize the design of the peptide linker in cp? using computational design methods;(3) Clone these new cp? variants into a coexpression vector that includes a covalently linked tandem repeat of human ?-globin ("di? globin");(4) Characterize the ligand binding and the structures of the mutant hemoglobins, by established spectroscopic methods (photolysis, stopped-flow, 1-D and 2-D 1H-NMR). Specific aims (1) and (3) will be carried out using standard gene cloning and/or synthesis with plasmids that are available from collaborators. Specific aim (2) will be achieved through collaboration with Prof. Brian Kuhlman (U. N. Carolina) who has expertise in the design of surface loops using RosettaDesign software. Our lab will then clone the gene(s) encoding cp? with the optimized linker sequence, and characterize the structure and function of the new mutant(s). Specific aim (4) will be carried out using specialized equipment in the lab of Prof. John Olson (Rice U.) to determine ligand binding characteristics of globin mutants by stopped-flow/flash photolysis, and equilibrium O2 binding. Our lab will characterize the structures of the mutant globins using near-UV circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. PUBLIC HEALTH RELEVANCE: In the 2005 Nationwide Blood Collection and Utilization Survey Report, the American Association of Blood Banks reported on the shrinking margin between the number of available units of blood approved for administration and the number of transfusions. The goal of this research is to address the projected shortfall in blood needed for transfusion, by developing a safe and efficacious red cell substitute based on modification of the human hemoglobin protein.