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.

Isothermal titration calorimetric and spectroscopic studies on (alcohol+salt) induced partially folded state of α-lactalbumin and its binding with 8-anilino-1-naphthalenesulfonic acid

“…Fluoro-substituted alcohols have been shown to exhibit structure stabilizing effects on proteins and peptides [13][14][15]. In recent years, use of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) has been reported in the generation of intermediate conformations and several other applications in biochemical studies [16][17][18][19][20][21]. The biological implication of HFIP has been reported in the investigation of prion diseases and Alzhiemer's amyloid peptides [22,23].…”

“…The biological implication of HFIP has been reported in the investigation of prion diseases and Alzhiemer's amyloid peptides [22,23]. It has been used to induce refolding in case of Cobra neurotoxin in the presence of sodium dodecyl sulphate [20]. HFIP has effectively formed aggregates in cecropin AD [19] and molten globule (MG) states in myoglobin [18].…”

Resolution of the effects induced by W → F substitutions on the conformation and dynamics of the amyloid-forming apomyoglobin mutant W7FW14F

“…F at either position 7 or 14 affects the protein conformation, dynamics, and stability at both pH 7.0 and 4.0. The latter condition favors the existence of a compact, partially folded intermediate formed by the A, G, and H helices and part of the B helix, with the remainder of the molecule more loosely packed with fluctuating D and E helices (Hughson et al 1990;Kay and Baldwin 1996;Barrick and Baldwin 1993;Sirangelo et al 1998Sirangelo et al , 2003a.…”

“…1). The folding of this protein is known to proceed through compact intermediates that have been detected in both kinetic and equilibrium experiments (Hughson et al 1990; Kay and Baldwin 1996; Barrick and Baldwin 1993;Sirangelo et al 1998Sirangelo et al , 2003a. In most of these intermediates, A, G, and H helices are folded and sterically oriented as in the native AGH subdomain, whereas the remainder of the molecule seems to be unordered.…”

4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization

“…Due to mixed hydrophilic-hydrophobic character, alcohols especially fluoro-substituted have been widely used to generate partially folded states in the proteins and peptide fragments with an aim to contribute to the area of protein folding and understanding of the conformation of proteins in aqueous organic solutions [1][2][3][4][5][6][7][8]. However, a study on the interaction of other halogen-substituted alcohols with proteins may also provide useful information on the conformation of proteins which could be linked to the intermediate states in the protein folding.…”