Daughdrill Gary W, Kashtanov Stepan, Stancik Amber, Hill Shannon E, Helms Gregory, Muschol Martin, Receveur-Bréchot Véronique, Ytreberg F Marty
Department of Cell Biology, Microbiology, and Molecular, University of South Florida, Tampa, FL 33612, USA.
Mol Biosyst. 2012 Jan;8(1):308-19. doi: 10.1039/c1mb05243h. Epub 2011 Oct 6.
Developing a comprehensive description of the equilibrium structural ensembles for intrinsically disordered proteins (IDPs) is essential to understanding their function. The p53 transactivation domain (p53TAD) is an IDP that interacts with multiple protein partners and contains numerous phosphorylation sites. Multiple techniques were used to investigate the equilibrium structural ensemble of p53TAD in its native and chemically unfolded states. The results from these experiments show that the native state of p53TAD has dimensions similar to a classical random coil while the chemically unfolded state is more extended. To investigate the molecular properties responsible for this behavior, a novel algorithm that generates diverse and unbiased structural ensembles of IDPs was developed. This algorithm was used to generate a large pool of plausible p53TAD structures that were reweighted to identify a subset of structures with the best fit to small angle X-ray scattering data. High weight structures in the native state ensemble show features that are localized to protein binding sites and regions with high proline content. The features localized to the protein binding sites are mostly eliminated in the chemically unfolded ensemble; while, the regions with high proline content remain relatively unaffected. Data from NMR experiments support these results, showing that residues from the protein binding sites experience larger environmental changes upon unfolding by urea than regions with high proline content. This behavior is consistent with the urea-induced exposure of nonpolar and aromatic side-chains in the protein binding sites that are partially excluded from solvent in the native state ensemble.
构建对内在无序蛋白（IDP）平衡结构集合的全面描述对于理解其功能至关重要。p53反式激活结构域（p53TAD）是一种与多种蛋白质伙伴相互作用且含有众多磷酸化位点的IDP。我们使用了多种技术来研究p53TAD在其天然状态和化学去折叠状态下的平衡结构集合。这些实验结果表明，p53TAD的天然状态尺寸类似于经典的无规卷曲，而化学去折叠状态则更为伸展。为了研究导致这种行为的分子特性，我们开发了一种生成IDP多样且无偏差结构集合的新算法。该算法用于生成大量合理的p53TAD结构，并重新加权以识别与小角X射线散射数据拟合最佳的结构子集。天然状态集合中的高权重结构显示出定位于蛋白质结合位点和脯氨酸含量高的区域的特征。定位于蛋白质结合位点的特征在化学去折叠集合中大多被消除；而脯氨酸含量高的区域则相对不受影响。核磁共振实验数据支持这些结果，表明与脯氨酸含量高的区域相比，蛋白质结合位点的残基在尿素诱导的去折叠过程中经历了更大的环境变化。这种行为与天然状态集合中部分被溶剂排斥的蛋白质结合位点中非极性和芳香族侧链的尿素诱导暴露一致。
