Pina David G, Gómez Javier, England Patrick, Craescu Constantin T, Johannes Ludger, Shnyrov Valery L
Laboratoire trafic et signalisation, UMR 144 CNRS, Institut Curie, 75248 Paris cedex 05, France.
Biochimie. 2006 Sep;88(9):1199-207. doi: 10.1016/j.biochi.2006.03.010. Epub 2006 Apr 25.
The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.
在平衡条件下，通过远紫外、近紫外圆二色光谱（CD）、内源荧光、分析型超速离心和差示扫描量热法（DSC），研究了2,2,2-三氟乙醇（TFE）浓度增加对志贺毒素B亚基（STxB）构象稳定性的影响。STxB是一种细菌同源五聚体蛋白，参与细胞表面结合和细胞内运输。我们的数据表明，TFE的存在会严重干扰STxB的天然结构。事实上，当TFE浓度高于20%（v/v）时，蛋白质的天然五聚体构象会协同转变为富含螺旋的单体和部分折叠的构象状态，且没有明显的三级结构。此外，当TFE浓度进一步增加（高于40%（v/v））时，未检测到协同转变。使用DSC和CD光谱在几种不同的TFE浓度下研究了STxB的热稳定性。在TFE浓度高达20%（v/v）时的热转变成功拟合到与蛋白质从五聚体折叠构象到单体状态转变相关的两态折叠/去折叠耦合寡聚化模型，TFE的存在将其稳定为部分折叠构象。
