Dissecting contributions to the denaturant sensitivities of proteins
Dissecting contributions to the denaturant sensitivities of proteins
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DOI：
10.1021/bi048389g
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发表时间：
2005-01-18
期刊：
影响因子：
2.9
通讯作者：
Mason, PE
中科院分区：
文献类型：
--
作者：
Dempsey, CE;Piggot, TJ;Mason, PE
了解尿素和氯化胍（GdmCl）使蛋白质变性的分子基础，应考虑到这样的观察结果，即在摩尔基础上，GdmCl作为蛋白质变性剂的有效性通常是尿素的2-2.5倍。先前的研究[Smith，J.S.，和Scholtz，JM。（1996）Biochemistry 35，7292-7297]已经表明，GdmCl对基于丙氨酸的螺旋肽的稳定性的影响可以分为变性剂和盐效应，因为向尿素中加入等摩尔NaCl将尿素诱导的解折叠增强到接近Gdm的程度。我们使用缺乏侧链静电对稳定性的贡献的基于丙氨酸的螺旋肽（alahel）重新研究了这一观察结果，并将该肽与色氨酸拉链的相对变性剂敏感性进行了比较。肽（trpzip），其天然构象主要通过交叉链吲哚环相互作用而稳定。与Smith和Scholtz的观察结果相反，GdmCl在无盐缓冲液中作为alahel的变性剂仅略强于尿素（变性剂m值m（GdmCl）/m（尿素）比= 1.4），并且尿素对alahel的变性仅表现出对NaCl或KCl的小依赖性。trpzip肽对GdmG比对尿素敏感得多（m（GdmCl）/m（尿素）= 3.5-4）。这些观察结果表明，蛋白质的m（GmCl）/m（尿素）比为2-2.5是对蛋白质稳定性的多重贡献的组合效应的结果，其中GdmCl可能仅比尿素稍微更有效（例如，氢键）或比脲有效得多（例如，吲哚-吲哚相互作用）。
Understanding the molecular basis for protein denaturation by urea and guanidinium chloride (GdmCl) should accommodate the observation that, on a molar basis, GdmCl is generally 2-2.5-fold more effective as a protein denaturant than urea. Previous studies [Smith, J. S., and Scholtz, J. M. (1996) Biochemistry 35, 7292-7297] have suggested that the effects of GdmCl on the stability of alanine-based helical peptides can be separated into denaturant and salt effects, since adding equimolar NaCl to urea enhanced urea-induced unfolding to an extent that was close to that of Gdm. We reinvestigated this observation using art alanine-based helical peptide (alahel) that lacks side chain electrostatic contributions to stability, and compared the relative denaturant sensitivities of this peptide with that of tryptophan zipper. peptides (trpzip) whose native conformations are stabilized largely by cross-strand indole ring interactions. In contrast to the observations of Smith and Scholtz, GdmCl was only slightly more powerful as a denaturant of alahel than urea in salt-free buffer (the denaturant m value m(GdmCl)/m(urea) ratio = 1.4), and the denaturation of alahel by urea exhibited only a small dependence on NaCl or KCl. The trpzip peptides were much more sensitive to GdmG than to urea (m(GdmCl)/m(urea) = 3.5-4). These observations indicate that the m(GmCl)/m(urea) ratio of 2-2.5 for proteins results from a combination of effects on the multiple contributions to protein stability, for which GdmCl may be only slightly more effective than urea (e.g., hydrogen bonds) or considerably more effective than urea (e.g., indole-indole interactions).