Cobos Eva S, Iglesias-Bexiga Manuel, Ruiz-Sanz Javier, Mateo Pedro L, Luque Irene, Martinez Jose C
Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, 18071 Granada, Spain.
Biochemistry. 2009 Sep 15;48(36):8712-20. doi: 10.1021/bi9007758.
WW domains are the smallest naturally independent beta-sheet protein structures available to date and constitute attractive model systems for investigating the determinants of beta-sheet folding and stability. Nonetheless, their small size and low cooperativity pose a difficult challenge for a quantitative analysis of the folding equilibrium. We describe here a comprehensive thermodynamic characterization of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) using a combination of calorimetric and spectroscopic techniques with several denaturing agents (temperature, pH, and chemical denaturants). Our results reveal that even though the experimental data can be described in terms of a two-state equilibrium, spectral data together with anomalous values for some thermodynamic parameters (a strikingly low temperature of maximum stability, a higher than expected native-state heat capacity, and a small specific enthalpy of unfolding) could be indicative of more complex types of equilibria, such as one-state downhill folding or alternative native conformations. Moreover, double-perturbation experiments reveal some features that, in spite of the apparent linear correlation between the thermodynamic parameters, seem to be indicative of a complex conformational equilibrium in the presence of urea. In summary, the data presented here point toward the existence of a low-energy barrier between the different macrostates of hNedd4-WW4, placing it at the frontier of cooperative folding.
WW结构域是迄今为止已知的最小的天然独立β-折叠蛋白质结构，是研究β-折叠的折叠和稳定性决定因素的极具吸引力的模型系统。尽管如此，它们的小尺寸和低协同性给折叠平衡的定量分析带来了艰巨挑战。我们在此描述了人类泛素连接酶Nedd4的第四个WW结构域（hNedd4-WW4）构象平衡的全面热力学特征，采用了量热法和光谱技术相结合的方法，并使用了几种变性剂（温度、pH值和化学变性剂）。我们的结果表明，尽管实验数据可以用两态平衡来描述，但光谱数据以及一些热力学参数的异常值（最大稳定性时温度极低、天然态热容高于预期、解折叠的比焓较小）可能表明存在更复杂的平衡类型，例如单态下坡折叠或替代天然构象。此外，双扰动实验揭示了一些特征，尽管热力学参数之间存在明显的线性相关性，但在尿素存在下似乎表明存在复杂的构象平衡。总之，本文提供的数据表明hNedd4-WW4的不同宏观状态之间存在低能垒，使其处于协同折叠的前沿。
