Sodiation of melittin, cytochrome c, and ubiquitin studied by electrospray mass spectrometry: Stabilities of α-helix and β-sheet in basic solutions

Metal ions play major roles in the functioning of biological systems. In our previous work, site-specific sodiation of gramicidin S (G), which is a cyclic antiparallel β-sheet consisting of four intramolecular N–H⋅⋅⋅OC hydrogen bonds, was examined. It was found that amide N–H bonds in [G + nH]ⁿ⁺ with n = 1 and 2 were deprotonated to form [G + nH − mH + mNa]ⁿ⁺ with m up to 7 and 8, respectively, in a 1 mM NaOH water/methanol (1/1) solution. In this work, sodiation of melittin (MEL) which is composed of two α-helices was studied. In a 1 mM NaOH solution, the maximum number of m for [MEL + nH − mH + mNa]ⁿ⁺ was found to increase with n, i.e., m = 4 for n = 2, m = 5 for n = 3, and m = 8 for n = 4. This suggests the occurrence of partial denaturation of the α-helix with an increase in n. The denaturation may be caused by loosening of intramolecular amide hydrogen bonds in the C-terminus side of MEL which is composed of 4 basic residues. For further investigation, sodiation of cytochrome c composed of α-helices, and ubiquitin composed of α-helices and a β-sheet was examined. In a 1 mM NaOH water/methanol solution, cytochrome c maintains its native conformation and only 9 acidic sites in addition to 15 carboxylic groups (total 24) are sodiated. This suggests that cytochrome c is resistive to sodiation in a 1 mM NaOH solution. In contrast to cytochrome c, denaturation of ubiquitin was observed in a 1 mM NaOH water/methanol solution. This suggests that β-sheet is less resistive to sodiation than α-helix. The denatured ubiquitin may have a molten globule conformation due to the presence of folded α-helices that act as hinges to prevent extensive unfolding.