title: Tryptophanyl contributions to apomyoglobin fluorescence resolved by site-directed mutagenesis.
abstract: The individual emission properties of the two tryptophanyl residues of sperm-whale apomyoglobin have been resolved by examining the fluorescence variations induced by denaturants, i.e., acid and guanidine, on apomyoglobin mutants W7F and W14F. The fluorescence changes have been correlated to the conformational transitions undergone by apomyoglobin on increasing denaturant concentration. The results indicate that the fluorescence decrease, observed for sperm-whale apomyoglobin on going from pH 8.0 to pH 6.0, cannot be ascribed to the formation of a charge transfer complex between a nearby histidine residue and W14 as reported in earlier papers but rather to minor structural changes affecting the microenvironments of both residues. The formation of the acidic partly folded state around pH 4.0 determines an increase of the fluorescence yield and a small red shift (5 nm) of W7 due to removal of sterically interacting K79, which is able to attenuate the emission of this residue in the native state. The fluorescence intensity of the other residue, i.e., W14, is not affected by the acidic transition. Guanidine denaturation experiments revealed an increase of fluorescence yield of W14 upon the intermediate formation, whereas the fluorescence of the other residue remained constant. The results suggest that the unfolding pathway may be different depending on the chemical nature of the denaturant used.
authors.affiliation: Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Via Costantinopoli 16, 80138, Naples, Italy.
authors.forename: I
authors.initials: I
authors.lastname: Sirangelo
authors.forename: S
authors.initials: S
authors.lastname: Tavassi
authors.forename: G
authors.initials: G
authors.lastname: Irace
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan
chemical_list: D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D014364:Tryptophan