Structural significance of the amino-terminal residues of sperm whale myoglobin

Abstract

Following the development of a nondestructive synthetic procedure for rapid production of des-Val1-myoglobin in large quantities, synthesis of a series of myoglobin derivatives varying in structure and charge in the NH2-terminal region was accomplished. In comparison to the untreated myoglobin, the des-Val1-myoglobin possessed at low pH a decreased stability and an increased net positive charge in the pH range 5.5-8.5. While the elevated net positive charge was no longer apparent after removal of the 2nd residue, instability of the molecule was sharply increased. Substitutions of the 1st residue, directed toward elucidating its structural importance, included glutamic acid, lysine and glycine. Addition of any of the 3 amino acids to the des-Val1-myoglobin restored much of the acid stability, with the [Gly1]myoglobin appearing nearly identical with the native molecule. All 3 semisynthetic myoglobins showed potentiometric titration curves characteristic of their respective, substituted residue. Carbamylation of the NH2 terminal of myoglobin and des-Val1-myoglobin yielded 2 nearly identical molecules in terms of all physical properties examined. It was concluded that the 1st residue primarily serves the function of maintaining the positively charged NH2 terminus a certain distance away from the beginning of the A helix and from the charge pair interaction of Lys-133 with Glu-6. Through physical measurements of the des-Val1,Leu2-myoglobin prior and subsequent to carbamylation of the NH2 terminus, it was apparent that the stabilization conferred on the des-Val1-myoglobin by the 2nd residue was dependent largely upon the hydrophobic interactions of its side chain.