{"title":"用电喷雾质谱法研究美乐汀、细胞色素 c 和泛素的钠化作用:α-螺旋和β-片在碱性溶液中的稳定性","authors":"Kenzo Hiraoka , Satoshi Ninomiya , Stephanie Rankin-Turner , Satoko Akashi","doi":"10.1016/j.ijms.2024.117212","DOIUrl":null,"url":null,"abstract":"<div><p>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⋅⋅⋅O<img>C hydrogen bonds, was examined. It was found that amide N–H bonds in [G + nH]<sup>n+</sup> with n = 1 and 2 were deprotonated to form [G + nH − mH + mNa]<sup>n+</sup> 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]<sup>n+</sup> 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 <em>c</em> 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 <em>c</em> maintains its native conformation and only 9 acidic sites in addition to 15 carboxylic groups (total 24) are sodiated. This suggests that cytochrome <em>c</em> is resistive to sodiation in a 1 mM NaOH solution. In contrast to cytochrome <em>c</em>, 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.</p></div>","PeriodicalId":338,"journal":{"name":"International Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":1.6000,"publicationDate":"2024-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Sodiation of melittin, cytochrome c, and ubiquitin studied by electrospray mass spectrometry: Stabilities of α-helix and β-sheet in basic solutions\",\"authors\":\"Kenzo Hiraoka , Satoshi Ninomiya , Stephanie Rankin-Turner , Satoko Akashi\",\"doi\":\"10.1016/j.ijms.2024.117212\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>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⋅⋅⋅O<img>C hydrogen bonds, was examined. It was found that amide N–H bonds in [G + nH]<sup>n+</sup> with n = 1 and 2 were deprotonated to form [G + nH − mH + mNa]<sup>n+</sup> 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]<sup>n+</sup> 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 <em>c</em> 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 <em>c</em> maintains its native conformation and only 9 acidic sites in addition to 15 carboxylic groups (total 24) are sodiated. This suggests that cytochrome <em>c</em> is resistive to sodiation in a 1 mM NaOH solution. In contrast to cytochrome <em>c</em>, 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. 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引用次数: 0
摘要
金属离子在生物系统的运作中发挥着重要作用。篦麻素 S(G)是一种由四个分子内 N-H⋅⋅OC 氢键组成的环状反平行 β-片材,我们在之前的工作中研究了篦麻素 S(G)的位点特异性钠化。研究发现,在 1 mM NaOH 水/甲醇(1/1)溶液中,n = 1 和 2 的 [G + nH]n+ 中的酰胺 N-H 键被去质子化,形成 m 分别高达 7 和 8 的 [G + nH - mH + mNa]n+。本研究对由两个 α-螺旋组成的美乐汀(MEL)的钠化作用进行了研究。在 1 mM NaOH 溶液中,发现[MEL + nH - mH + mNa]n+ 的最大 m 数随 n 的增加而增加,即 n = 2 时 m = 4,n = 3 时 m = 5,n = 4 时 m = 8。变性可能是由 4 个碱性残基组成的 MEL 的 C 端分子内酰胺氢键松动所致。为了进一步研究,研究人员对由α-螺旋组成的细胞色素 c 和由α-螺旋和β-片组成的泛素进行了碱化。在 1 mM NaOH 水/甲醇溶液中,细胞色素 c 保持原生构象,除 15 个羧基(共 24 个)外,只有 9 个酸性位点被钠化。这表明细胞色素 c 在 1 mM NaOH 溶液中具有抗碱化性。与细胞色素 c 相反,在 1 mM NaOH 水/甲醇溶液中观察到泛素变性。这表明,β-片状结构比α-螺旋结构对碱化的抵抗力更弱。变性后的泛素可能具有熔融球状构象,这是因为存在折叠的 α-螺旋,它们起着铰链的作用,防止广泛的解折。
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.
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The journal invites papers that advance the field of mass spectrometry by exploring fundamental aspects of ion processes using both the experimental and theoretical approaches, developing new instrumentation and experimental strategies for chemical analysis using mass spectrometry, developing new computational strategies for data interpretation and integration, reporting new applications of mass spectrometry and hyphenated techniques in biology, chemistry, geology, and physics.
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