The effect of tryptophanyl substitution on folding and structure of myoglobin.
Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A-5) and 14 (A-12) in the N-terminal region (A helix) of the protein molecule. The crucial role of tryptophanyl residues has been investigated by site-directed mutagenesis and molecular dynamics simulation. The apomyoglobin mutants with a double W-->F substitution were found to be not correctly folded and therefore not expressed as holoprotein. The introduction of a tyrosyl residue at position 7, that is, W7YW14F, resulted in the expression of a correctly folded myoglobin. Not correctly folded apomyoglobins were found with the following mutants: W7FW14Y, W7EW14F, W7FW14E, W7KW14F, W7FW14K. Moreover, in all these cases, very low levels of expression were observed. The acid-induced denaturation curves of wild-type and folded mutant W7YW14F, obtained following the fluorescence variation of the extrinsic fluorophore 1-anilino-8-naphthalenesulfonate, revealed that the stability of the native state of mutant apoprotein is decreased, thus indicating that the replacement W-->Y in position 7 is able to restore a correct folding but not the same stability. Molecular dynamics simulation indicated that both tryptophans are involved in forming favorable, specific tertiary interactions in the native apomyoglobin structure. The lack of some of these interactions caused by tryptophanyl replacement affects the overall protein structure and may provide an explanation for the observed stability decrease. In the case of the double W-->F substitution, the simulated structure shows conclusively the domain formed by helices A, G and H to be not correctly folded. This effect is attenuated if at least one of the two residues is conserved or a tyrosyl residue replaces W7.
Sirangelo
I
I
Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Italy.
Tavassi
S
S
Martelli
P L
PL
Casadio
R
R
Irace
G
G
D009211:Myoglobin / Q000737:chemistry*; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry
D009211:Myoglobin; D014364:Tryptophan