Sathish H A, Cusan Monica, Aisenbrey Christopher, Bechinger Burkhard
Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany.
Biochemistry. 2002 Apr 30;41(17):5340-7. doi: 10.1021/bi0115784.
The conformational stabilities of full-length colicin B and its isolated C-terminal domain were studied by guanidine hydrochloride induced unfolding. The unfolding/refolding was monitored by far-UV CD and intrinsic tryptophan fluorescence spectroscopies. At pH 7.4, the disruption of the secondary structure of full-length colicin B is monophasic, while changes in tertiary structure occur in two separate transitions. The intermediate species, which is well-populated around 2.2 M guanidine hydrochloride, exhibits secondary and tertiary structures distinct from both native and unfolded states. Whereas the domain structure of native full-length colicin B is reflected in its DSC profile, the folding intermediate of the same protein exhibits a single unresolved peak. These observations have led us to propose an unfolding model for full-length colicin B where the first transition between 0 and 2.5 M GuHCl with an associated free energy of 3 kcal/mol correlates with the partial unfolding of the R/T domain. The stability of full-length colicin B is weakened due to the presence of the R/T domain in both the native [Ortega, A., Lambotte, S., and Bechinger, B. (2001) J. Biol. Chem. 276 (17), 13563-13572] and the intermediate states. The second transition between 2.5 and 5 M GuHCl involves unfolding of the C-terminal domain (Delta = 7 kcal/mol). The isolated colicin B C-terminal domain consists of two subdomains, and the two parts of this protein fragment unfold sequentially through the formation of at least one intermediate. The significance of these results for membrane insertion of colicin B is discussed.
通过盐酸胍诱导的解折叠研究了全长大肠杆菌素B及其分离的C末端结构域的构象稳定性。通过远紫外圆二色光谱和内在色氨酸荧光光谱监测解折叠/重折叠过程。在pH 7.4时，全长大肠杆菌素B二级结构的破坏是单相的，而三级结构的变化发生在两个独立的转变过程中。在约2.2 M盐酸胍浓度下大量存在的中间物种，其二级和三级结构与天然态和未折叠态均不同。天然全长大肠杆菌素B的结构域结构反映在其差示扫描量热曲线中，而同一蛋白质的折叠中间体呈现出一个未解析的单峰。这些观察结果使我们提出了全长大肠杆菌素B的解折叠模型，其中在0至2.5 M盐酸胍之间的第一个转变以及相关的3 kcal/mol的自由能变化与R/T结构域的部分解折叠相关。由于R/T结构域在天然态[奥尔特加，A.，兰博特，S.，和贝兴格，B.（2001）《生物化学杂志》276（17），13563 - 13572]和中间态中均存在，全长大肠杆菌素B的稳定性被削弱。在2.5至5 M盐酸胍之间的第二个转变涉及C末端结构域的解折叠（Δ = 7 kcal/mol）。分离的大肠杆菌素B C末端结构域由两个亚结构域组成，并且该蛋白质片段的两个部分通过形成至少一种中间体依次解折叠。讨论了这些结果对大肠杆菌素B膜插入的意义。
