Resolution of tryptophan-ANS fluorescence energy transfer in apomyoglobin by site-directed mutagenesis.
Resonance energy transfer between tryptophanyl residues and the apolar fluorescent dye 1-anilino-8-naphthalene sulfonate (ANS) occurs when the fluorophore is bound to native folded sperm whale apomyoglobin. The individual transfer contribution of the two tryptophanyl residues (W7 and W14, both located on the A-helix of the protein) was resolved by measuring the tryptophan-ANS transfer efficiency for the ANS-apomyoglobin complexes formed by wild-type protein and protein mutants containing one or no tryptophanyl residues, i.e. W7F, W14F and W7YW14F. The transfer efficiency of W14 residue was found to be higher than that of W7, thus indicating that W14 acts as the main energy donor in the ANS-apomyoglobin complex. This suggests that the plane containing the anilinonaphthalene ring of the extrinsic fluorophore has a spatial orientation similar to that of W14 and, hence, to the heme group in the holoprotein.
Sirangelo
Ivana
I
Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Italy.
Malmo
Clorinda
C
Casillo
Mariateresa
M
Irace
Gaetano
G
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry*; D014907:Whales
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D005456:Fluorescent Dyes; D009211:Myoglobin; C010857:apomyoglobin; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan