title: Hexafluoroisopropanol and acid destabilized forms of apomyoglobin exhibit structural differences.
abstract: The conformational properties of partially folded states of apomyoglobin have been investigated using an integrated approach based on fluorescence spectroscopy and hydrogen/deuterium exchange followed by mass spectrometry. The examined states were those obtained: (i) by adding 4% v/v hexafluoroisopropanol to native myoglobin, HFIP-MG(N); (ii) by adding 4% v/v hexafluoroisopropanol to acid unfolded myoglobin, HFIP-MG(U); (iii) at pH 3.8, I-1 state; and (iv) at pH 2.0-0.2 M NaCl, A state. Proteolytic digestion of the hydrogen/deuterium exchanged proteins showed that, in I-1 state, the helices C, D, E, and F incorporate more deuterium, whereas in HFIP-MG(N) the exchange rate is similar for all protein regions. These results suggest that I-1 contains the ABGH domain in a native-like organization, whereas HFIP-MG(N) loses a large number of tertiary interactions, thus acquiring a more flexible structure. The fluorescence data are consistent with the above picture. In fact, the tryptophan/ANS energy transfer is much less efficient for the ANS-HFIP-MG(N) complex than for the other complexes, thus suggesting that the distances between the fluorophores might be increased. Moreover, fluorescence polarization measurements indicated that the rotational motion of HFIP-MG(N) occurs on a longer time scale than the other partially folded states, thus suggesting that the volume of this state could be larger. The overall results indicate that addition of hexafluoroisopropanol to native myoglobin results in the formation of a true molten globule where tertiary interactions are reduced, while the secondary structure and the globular compactness are conserved.
authors.affiliation: Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy.
authors.forename: Ivana
authors.initials: I
authors.lastname: Sirangelo
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Dal Piaz
authors.forename: Clorinda
authors.initials: C
authors.lastname: Malmo
authors.forename: Mariateresa
authors.initials: M
authors.lastname: Casillo
authors.forename: Leila
authors.initials: L
authors.lastname: Birolo
authors.forename: Piero
authors.initials: P
authors.lastname: Pucci
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marino
authors.forename: Gaetano
authors.initials: G
authors.lastname: Irace
mesh_terms: D000143:Acids; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D017666:Deuterium Oxide; D006736:Horses; D006863:Hydrogen-Ion Concentration; D009211:Myoglobin / Q000737:chemistry*; D020005:Propanols; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011522:Protons; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D006845:Hydrocarbons, Fluorinated
chemical_list: D000143:Acids; D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D017666:Deuterium Oxide; D009211:Myoglobin; D020005:Propanols; D011522:Protons; C010857:apomyoglobin; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C027132:1-anilino-8-naphthalenesulfonate