Resolution of the effects induced by W → F substitutions on the conformation and dynamics of the amyloid-forming apomyoglobin mutant W7FW14F.
Myoglobin is an alpha-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 increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.
Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Naples, Italy.
Giuseppe
G
Infusini
Clara
C
Iannuzzi
Silvia
S
Vilasi
Leila
L
Birolo
Daniela
D
Pagnozzi
Piero
P
Pucci
Gaetano
G
Irace
Ivana
I
Sirangelo
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D013057:Spectrum Analysis; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014907:Whales
D000682:Amyloid; D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D010649:Phenylalanine; D014364:Tryptophan