W-F substitutions in apomyoglobin increase the local flexibility of the N-terminal region causing amyloid aggregation: a H/D exchange study.
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 the 12-29 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.
Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Via L. de Crecchio 7, Naples, 80138. Italy.
Giuseppe
G
Infusini
Clara
C
Iannuzzi
Silvia
S
Vilasi
Rosa
R
Maritato
Leila
L
Birolo
Daniela
D
Pagnozzi
Piero
P
Pucci
Gaetano
G
Irace
Ivana
I
Sirangelo
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D059748:Proteolysis
D000682:Amyloid; D001059:Apoproteins; D050505:Mutant Proteins; D009211:Myoglobin; D010446:Peptide Fragments; C010857:apomyoglobin