The N-terminal sequence affects distant helix interactions in hemoglobin -Implications for mutant proteins from studies on recombinant hemoglobin felix

The N-terminal 18-amino acid sequence of the beta-chain of hemoglobin, as f ar as the end of the A helix, has been replaced by the corresponding sequen ce of the gamma-chain of fetal hemoglobin with the remaining sequence of th e beta-chain retained (helices B through H), The gamma-beta-chain had the c orrect mass, and its entire sequence was established by mass spectrometric analysis of its tryptic peptides; the alpha-chain also had the correct mass . This recombinant hemoglobin (named Hb Felix) retains cooperativity and ha s an oxygen affinity like that of HbA both in the presence and absence of t he allosteric regulators, 2,3-diphosphoglycerate or chloride but differs fr om HbF in its 2,3-diphosphoglycerate response. However, Hb Felix has some f eatures that resemble fetal hemoglobin, i.e. its significantly decreased te tramer-dimer dissociation and its circular dichroism spectrum, which measur e the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively. Even though Hb F elix contains the HbA amino acids at its tetramer-dimer interface, which is located at a distance from the substitution sites, its interface propertie s resemble those of HbF, Therefore, the N-terminal sequence and not just th ose amino acids directly involved at the subunit interface contacts with al pha-chains must have a strong influence on this region of the molecule. The results reinforce the concept of fluid long range relationships among vari ous parts of the hemoglobin tetramer (Dumoulin, A. Manning, L. R., Jenkins, W. T., Winslow, R. M., and Manning, J. M. (1997) J, Biol. Chem. 272, 31326 -31332) and demonstrate the importance of the N-terminal sequence, especial ly in some mutant hemoglobins, in influencing its overall structure by affe cting the relationship between helices.