The effects of amino acid substitution on apomyoglobin stability, folding intermediates, and holoprotein expression

Abstract

The stabilities and expression levels of over 150 randomly-generated triple mutants and 100 different rationally constructed single, double, and triple mutants of sperm whale myoglobin have been examined. The effects of mutagenesis on stability were quantified at 15 different positions in the heme pocket. The largest effects were seen at the internal residues E7 and E11, where large, apolar residues increase the overall stability of the apoglobin, and at the external residue E10, where residues with favorable helix propensities increase stability. Substitutions at other positions in the heme pocket also have significant effects on apoglobin stability. Multiple regression analyses were performed correlating overall stability with the hydrophobicity, size, and helix propensity of the replacement amino acid. In most cases, once a range of amino acids has been correlated at a given position, the stabilities of untested mutants can be predicted based on the hydropathy, size, and helix propensity of the substituted amino acid. More importantly, the stabilities of multiple mutants can be predicted from the measured unfolding constants of the corresponding single mutants or from the amino acid properties of the substituted amino acid using the regression analysis performed on the single mutants. Previous work has suggested a connection between apomyoglobin stability and expression. Our correlations with apoglobin unfolding constants show quantitatively that high stability is a prerequisite for high expression in E. coli, and that the stability of the apomyoglobin can explain over 50% of the variance in expression levels of all the mutants that have been examined. In addition, the potential expression levels for multiple mutants of myoglobin can be predicted from either the stabilities of the corresponding single mutants, or from the properties of the replacement amino acid, even if the stabilities have not been measured previously.