Eftink M R, Helton K J, Beavers A, Ramsay G D
Department of Chemistry, University of Mississippi, University 38677.
Biochemistry. 1994 Aug 30;33(34):10220-8. doi: 10.1021/bi00200a002.
The urea-induced unfolding of trp aporepressor from Escherichia coli has been studied as a function of pH from 2.5 to 12.0 at 25 degrees C. At pH 7 and above, the unfolding transition, as monitored by changes in the fluorescence intensity at 360 nm, shows a single transition. At low pH, the transition again appears to be a single transition. In the range of 3.5-6.0, the transition is biphasic, indicating the existence of a folding intermediate. The transitions have also been studied using circular dichroism and size exclusion chromatography. The data were fitted by a model in which the dimeric protein first unfolds to form structured monomers, followed by the unfolding of the monomers. From fits with this "folded monomers" model, the free energy change for the dimer<-->monomer dissociation becomes less positive as pH is decreased; the free energy change for the unfolding of the monomers is essentially independent of pH. An alternate model is one in which the dimer first undergoes a transition to a partially unfolded dimeric state, with this intermediate then denaturing to unfolded monomers. Both models give adequate fits to the data obtained at a single protein concentration. From a study of the concentration dependence of the urea-induced unfolding at pH 5, the "folded monomers" model is found to be more consistent with the data. Size exclusion chromatography data support the description of the intermediate state, which is the most populated state at low pH in the absence of urea, as being a relatively compact monomer.(ABSTRACT TRUNCATED AT 250 WORDS)
在25℃下，研究了尿素诱导的大肠杆菌色氨酸阻遏蛋白的去折叠过程与pH值（2.5至12.0）的关系。在pH 7及以上时，通过监测360nm处荧光强度的变化，去折叠转变呈现单一转变。在低pH值时，该转变似乎也是单一转变。在3.5 - 6.0范围内，转变是双相的，表明存在折叠中间体。还使用圆二色性和尺寸排阻色谱法研究了这些转变。数据通过一个模型进行拟合，在该模型中，二聚体蛋白首先去折叠形成结构化单体，随后单体去折叠。根据与这个“折叠单体”模型的拟合，随着pH值降低，二聚体⇄单体解离的自由能变化变得不那么正值；单体去折叠的自由能变化基本与pH值无关。另一种模型是二聚体首先转变为部分去折叠的二聚体状态，然后该中间体变性为去折叠单体。两种模型都能很好地拟合在单一蛋白质浓度下获得的数据。通过研究pH 5时尿素诱导去折叠的浓度依赖性，发现“折叠单体”模型与数据更一致。尺寸排阻色谱数据支持了中间态的描述，即在没有尿素的情况下，中间态是低pH时最主要的状态，是一种相对紧密的单体。
