Methanol-Induced Conformations of Myoglobin at pH 4.0

Abstract

The equilibrium methanol-induced conformation changes of holomyoglobin (hMb) at pH 4.0 have been studied by circular dichroism, tryptophan fluorescence, and Soret band absorption and by electrospray ionization mass spectrometry (ESI-MS). Optical spectra show the following: (1) In 35-40% (v/v) methanol/water, the native-like secondary structure remains, the tertiary structure is lost, the heme protein interactions are decreased, and a folding intermediate is formed. (2) In 50% methanol, heme is lost from the protein, and there is a small decrease in helicity together with a loss of tertiary structure. (3) At >60% methanol, the helicity increases and the apoprotein goes into a helical denatured state. The conformations are also probed by the charge states produced in ESI-MS and by hydrogen/deuterium (H/D) exchange with mass measurement by ESI-MS. At 0-30% methanol, native hMb produces relatively low charge states (9(+)-13(+)) in ESI-MS and exchanges relatively few hydrogens. In 35-40% methanol, at which an intermediate is formed, there is a bimodal distribution of hMb ions with both low (9(+)-13(+)) and high (14(+)-23(+)) charge states and also a high charge state distribution (12(+)-26(+)) of apomyoglobin (aMb) ions. Low and high charge states of hMb and a high charge state of aMb all show the same H/D exchange rate, indicating that an unfolded hMb intermediate interconverts between folded hMb and unfolded aMb. The charge state distribution for the unfolded hMb intermediate observed here is similar to that of the recently reported transient intermediate formed during the acid denaturation of hMb. At 50% alcohol the protein produces predominantly high charge states of aMb ions and shows H/D exchange rates close to those of the acid-denatured protein. H/D exchange of the helical denatured protein at alcohol concentrations >60%, at which high charge states of aMb are produced, shows that the protein structure is more protected than at approximately 50% methanol.

Tryptophanyl Substitutions in Apomyoglobin Determine Protein Aggregation and Amyloid-like Fibril Formation at Physiological pH

“…At least three partially folded intermediates, which differ in the amount of their secondary structure, have been detected at low pH in the presence of different anions (18 -21). Similar results were obtained in studies of methanol-induced conformational transitions (22). In these intermediates, A, G, and H helices are folded as in the native state, whereas the remainder of the molecule seems to be unordered.…”

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

“…First, the 2-propanol solvent molecules, present at high concentration in the crystallization medium, may serve as ligands of adequate polarity for the amino acid residues on that specific surface of the subunit that otherwise generally participates in subunit interactions. In agreement with this possibility, alcohols are reported to modulate protein folding (66,67). The fact that in the human enzyme crystal structure the protein still forms a trimer may be due to the differences in the crystallization condition (the human enzyme was precipitated with sodium citrate at pH 7.5-8.5).…”

Conformations of gas-phase ions of ubiquitin, cytochrome c, apomyoglobin, and β-lactoglobulin produced from two different solution conformations

“…Because of their lower H/D exchange levels in solution, the H c states are sometimes called the helical denatured "protected" states. The H and H c states both produce high charge state distributions in ESI, similar to the high charge state distributions produced by acid denatured proteins [9,10]. In this case, a direct comparison of the properties of the gas-phase protein ions produced from two different solution conformations is possible.…”

“…In 90% alcohol, proteins can form helical denatured states (termed "H c " states). The helical denatured states have higher ␣-helix content (up to 90% to 100%), distinct CD spectra, and lower solution H/D exchange levels than those of the H state and acid or urea denatured conformations [5][6][7][8][9][10]. Because of their lower H/D exchange levels in solution, the H c states are sometimes called the helical denatured "protected" states.…”