Steer B A, Merrill A R
Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
Biochemistry. 1997 Mar 11;36(10):3037-46. doi: 10.1021/bi961926f.
The equilibrium unfolding pathway of the colicin E1 channel peptide was shown in a previous study to involve an unfolding intermediate, stable in approximately 4 M guanidine hydrochloride, which comprised primarily the C-terminal hydrophobic alpha-helical hairpin segment of the peptide [Steer, B. A., & Merrill, A. R. (1995) Biochemistry 34, 7225-7233]. In this study, the structural nature of this unfolding intermediate was investigated further, and it was found that the intermediate primarily consists of a dimer species and is comprised of two partially denatured monomeric peptides, which appear to be associated by hydrophobic interactions. The dimerized structure was detected by size-exclusion high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical cross-linking, and intermolecular fluorescence energy transfer. Using stopped-flow fluorescence spectroscopy, the kinetics of the denaturation and dimerization of the colicin E1 channel peptide in 4 M guanidine hydrochloride were examined. Denaturation kinetics were also investigated by wild-type peptide Trp fluorescence and 1-anilinonaphthalene-8-sulfonic acid binding. The kinetics of dimer formation were examined by monitoring the time dependence of intermolecular Trp to 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid fluorescence resonance energy transfer upon denaturation in 4 M guanidine hydrochloride. In addition, single Trp mutant peptides were employed as site-specific fluorescent probes of unfolding kinetics and reported diverse and characteristic unfolding kinetics. However, it was shown that following a rapid and major unfolding transition the peptide's core residues cluster slowly, by hydrophobic association, forming an intermediate species which is a prerequisite to dimerization. These equilibrium and kinetic unfolding data describe a unique unfolding mechanism where the channel peptide forms a partially unfolded dimerized structure in 4 M guanidine hydrochloride.
在先前的一项研究中表明，大肠杆菌素E1通道肽的平衡去折叠途径涉及一种去折叠中间体，该中间体在约4M盐酸胍中稳定，主要由肽的C端疏水α-螺旋发夹片段组成[Steer, B. A., & Merrill, A. R. (1995) Biochemistry 34, 7225 - 7233]。在本研究中，对该去折叠中间体的结构性质进行了进一步研究，发现该中间体主要由二聚体物种组成，由两个部分变性的单体肽组成，它们似乎通过疏水相互作用缔合。通过尺寸排阻高效液相色谱、十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳、化学交联和分子间荧光能量转移检测到二聚化结构。使用停流荧光光谱法，研究了大肠杆菌素E1通道肽在4M盐酸胍中的变性和二聚化动力学。还通过野生型肽色氨酸荧光和1 - 苯胺基萘 - 8 - 磺酸结合研究了变性动力学。通过监测在4M盐酸胍中变性时分子间色氨酸到5 - [[2 - [(碘乙酰基)氨基]乙基]氨基]萘 - 1 - 磺酸荧光共振能量转移的时间依赖性来研究二聚体形成的动力学。此外，单色氨酸突变肽被用作去折叠动力学的位点特异性荧光探针，并报告了不同的特征性去折叠动力学。然而，结果表明，在快速且主要的去折叠转变之后，肽的核心残基通过疏水缔合缓慢聚集，形成一种中间体物种，这是二聚化的先决条件。这些平衡和动力学去折叠数据描述了一种独特的去折叠机制，其中通道肽在4M盐酸胍中形成部分去折叠的二聚化结构。
