The objective of this proposal is to develop chemical techniques for the enrichment of myoglobins, hemoglobins and other proteins with 13C or alternative nuclei suitable for NMR study. The methodology is aimed at the size of molecule for which total chemical synthesis of the polypeptide with a variety of enrichment sites is clearly impractical at the present time. The strategy, therefore, is to modify the natural protein by appropriate cleavage and resynthesis with the incorporation of the enriched amino acids by way of substitution of a fragment into the sequence. This approach has been termed semisynthesis. The chemical substitution methods will clearly serve a still broader objective, the substitution of alternative residues at given sequence positions to test structure-function relationships, to interconvert species specificity, or to redirect function. This work can be applied directly to understanding hemoglobin function and its control, and will form a basis for such broad applications as enzyme or hormone replacement therapy. In particular, the application aims at the detailed analysis by 13C NMR of enriched semisynthetic products in which residues are replaced in the initial 14 amino acids of the myoglobin chain. The analysis will deal with the motional behavior, electrostatic interactions and contributions to stability of residues in this segment of the molecule. Experiments to extend the methods to other parts of the molecule will be undertaken. The methods will be extended to human hemoglobin to encompass at first the enrichment of the 4 N-terminal valine residues for studies of effector interactions. Leading sequences will be attached to the N-terminus of myoglobin to test the requirements for anchoring in lipid bilayers among other questions involving hydrophobic interactions. Semisynthetic manipulation of the N-terminal segment of bovine pancreatic trypsin inhibitor will be made to evaluate electrostatic and other factors in the structure and function of this molecule. The experimental manipulations involved in this work meet the requirements for proteins and their fragments that must be handled in relatively polar solvents to avoid irreversible denaturation, and thus are of potentially wide application to other proteins and peptides.
