Wang Yuanyuan, Trewhella Jill, Goldenberg David P
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850, USA.
J Mol Biol. 2008 Apr 11;377(5):1576-92. doi: 10.1016/j.jmb.2008.02.009. Epub 2008 Feb 14.
The disulfide-reduced form of bovine ribonuclease A, with the Cys thiols irreversibly blocked, was characterized by small-angle x-ray scattering. To help resolve the conflicting results and interpretations from previous studies of this model unfolded protein, we measured scattering profiles using a range of solution conditions and compared them with the profiles predicted by a computational model for a random-coil polypeptide. Analysis of the simulated and experimental profiles reveals that scattering intensities at intermediate angles, corresponding to interatomic distances in the range of 5-20 A, are particularly sensitive to changes in solvation and can be used to assess the internal scaling behavior of the polypeptide chain, expressed as a mass fractal dimension, D(m). This region of the scattering curve is also much less sensitive to experimental artifacts than is the very small angle regime (the Guinier region) that has been more typically used to characterize unfolded proteins. The experimental small-angle x-ray scattering profiles closely matched those predicted by the computational model assuming relatively small solvation energies. The scaling behavior of the polypeptide approaches that of a well-solvated polymer under conditions where it has a large net charge and at high urea concentrations. At lower urea concentrations and neutral pH, the behavior of the chain approaches that expected for theta-conditions, where the effects of slightly unfavorable interactions with solvent balance those of excluded volume, leading to scaling behavior comparable to that of an idealized random walk chain. Though detectable, the shift toward more compact conformations at lower urea concentrations does not correspond to a transition to a globule state and is associated with little or no reduction in conformational entropy. This type of collapse, therefore, is unlikely to greatly reduce the conformational search for the native state.
牛核糖核酸酶A的二硫键还原形式，其半胱氨酸硫醇被不可逆地阻断，通过小角X射线散射进行表征。为了帮助解决此前对这种模型展开蛋白研究中相互矛盾的结果和解释，我们在一系列溶液条件下测量了散射曲线，并将其与随机卷曲多肽计算模型预测的曲线进行比较。对模拟曲线和实验曲线的分析表明，对应于5-20埃范围内原子间距离的中间角度处的散射强度，对溶剂化变化特别敏感，可用于评估多肽链的内部标度行为，用质量分形维数D(m)表示。与更常用于表征展开蛋白的极小角度区域（吉尼尔区域）相比，散射曲线的这个区域对实验假象也不那么敏感。实验性小角X射线散射曲线与假设相对较小溶剂化能的计算模型预测的曲线紧密匹配。在具有大量净电荷的条件下以及高尿素浓度下，多肽的标度行为接近良溶剂化聚合物的标度行为。在较低尿素浓度和中性pH值下，链的行为接近θ条件下预期的行为，即与溶剂的轻微不利相互作用的影响与排除体积的影响相平衡，导致标度行为与理想化随机游走链相当。尽管在较低尿素浓度下向更紧凑构象的转变是可检测的，但这并不对应于向球状状态的转变，并且与构象熵的减少很少或没有减少相关。因此，这种类型的塌缩不太可能大大减少对天然状态的构象搜索。
