Myoglobin is an α-helical globular protein containing two highly conserved tryptophanyl residues at positions 7
and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the
protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room
temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing
structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and
the amyloid forming mutant W7FW14F.
In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed
by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in
an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of
the12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the
formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation
at physiological pH.