This study proposes to utilize the current knowledge of hemoglobin science and genetic engineering technology to design and produce site-specific mutants of human hemoglobin in order to gain new insights into the dioxygen binding properties of hemoglobin. The theoretical and technical results obtained from these studies could be usefully applied to studies of structure-function of other proteins. In this study, oligonucleotides will be designed and synthesized to perform site-directed mutagenesis of normal human globin genes. Site-specific modified globins will be expressed in E. coli by a cleavable fusion protein expression vector containing the newly constructed mutant gene. After purification, the mutant globin will be liberated from the fusion protein by an enzymatic cleavage. The mutant globin will then be renatured and reconstituted with heme and the partner chain to form a tetrameric hemoglobin. After further purification, the product will be used for detailed functional analysis to pursue the primary goal of structure-function studies and for other collaborative studies in the fields of molecular biology, biophysics, and biochemistry. A combination of studies of naturally occurring human variant, and chemically modified hemoglobins will lead to better understanding of the functional geometry of human normal hemoglobin. Hypotheses that involve structural influences on functional and physicochemical properties of the molecule will be tested. Further design and construction of model hemoglobins with predictable novel properties will be attempted.
| Perutz, M F; Shih, D T; Williamson, D (1994) The chloride effect in human haemoglobin. A new kind of allosteric mechanism. J Mol Biol 239:555-60 |
| Shih, D T; Luisi, B F; Miyazaki, G et al. (1993) A mutagenic study of the allosteric linkage of His(HC3)146 beta in haemoglobin. J Mol Biol 230:1291-6 |
| Fermi, G; Perutz, M F; Williamson, D et al. (1992) Structure-function relationships in the low-affinity mutant haemoglobin Aalborg (Gly74 (E18)beta----Arg). J Mol Biol 226:883-8 |
| Bonaventura, C; Cashon, R; Bonaventura, J et al. (1991) Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated beta chains. J Biol Chem 266:23033-40 |
