Schönbrunner N, Wey J, Engels J, Georg H, Kiefhaber T
Biozentrum der Universität Basel, Abteilung Biophysikalische Chemie, Switzerland.
J Mol Biol. 1996 Jul 19;260(3):432-45. doi: 10.1006/jmbi.1996.0412.
The effect of trifluoroethanol (TFE) on the structure of the all-beta-sheet protein tendamistat was investigated. At low concentrations TFE induces cooperative loss of the native tertiary structure leading to a partially folded state. The loss of specific side-chain interactions in the transition from the native state of the TFE-induced state is demonstrated by the disappearance of the CD bands in the aromatic region, a reduced chemical shift dispersion of the one-dimensional 1H NMR spectrum and a broad, uncooperative thermal unfolding transition of the partially folded state. An increased line-width of the NMR bands in the TFE state compared with the unfolded state suggests the presence of multiple, rapidly interconverting conformations. Hydrogen-exchange studies of amide proteins in the TFE state reveal the existence of defined hydrogen bonds at the same locations as in the native state, but with largely reduced stability. This suggests the presence of most of the native beta-sheet structure. These results are supported by Fourier transformed IR measurements, which show nearly the same amount of beta-structure in the TFE state and in the native state. Far UV CD spectroscopy suggests the induction of some alpha-helical structure upon addition of TFE, which appears to be located mainly in regions corresponding to loops or random structure in the native state and which seems to represent fluctuating conformations with preferred backbone angles rather than stable, hydrogen-bonded alpha-helices. These results show that stable non-local interactions, as they occur in beta-sheets, can form in the absence of specific side-chain interactions. The presence of a subset of the native long-range interactions and the absence of stable non-native interactions suggests that the observed partially folded state might represent an early intermediate on a hierarchical folding pathway of tendamistat.
研究了三氟乙醇（TFE）对全β-折叠蛋白腱糖胺聚糖结构的影响。在低浓度下，TFE诱导天然三级结构的协同丧失，导致部分折叠状态。从天然状态转变为TFE诱导状态时特定侧链相互作用的丧失，通过芳香区CD谱带的消失、一维¹H NMR谱化学位移分散的降低以及部分折叠状态的宽且不协同的热解折叠转变得以证明。与未折叠状态相比，TFE状态下NMR谱带的线宽增加，表明存在多种快速相互转化的构象。对TFE状态下酰胺蛋白的氢交换研究表明，在与天然状态相同的位置存在确定的氢键，但稳定性大大降低。这表明存在大部分天然β-折叠结构。傅里叶变换红外测量结果支持了这些结果，该测量显示TFE状态和天然状态下β-结构的量几乎相同。远紫外CD光谱表明，加入TFE后会诱导一些α-螺旋结构，这些结构似乎主要位于天然状态下对应于环或无规结构的区域，并且似乎代表具有优选主链角度的波动构象，而不是稳定的、氢键结合的α-螺旋。这些结果表明，如在β-折叠中发生的稳定非局部相互作用可以在没有特定侧链相互作用的情况下形成。天然远程相互作用子集的存在和稳定非天然相互作用的缺失表明，观察到的部分折叠状态可能代表腱糖胺聚糖分级折叠途径上的早期中间体。
