Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal.
iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901, Oeiras, Portugal.
Sci Rep. 2019 Feb 7;9(1):1647. doi: 10.1038/s41598-018-37599-z.
Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established H-N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.
理解蛋白质结构和动力学，这些结构和动力学控制着关键的细胞过程，对于基础研究和应用研究至关重要。固有无序蛋白质（IDP）区域通过交替的瞬态构象显示多功能性，是疾病机制中的关键参与者。IDP 区域非常丰富，尤其是在小型病毒中，允许在小蛋白质组中实现大量功能。因此，蛋白质功能与结构之间的关系现在从一个不同的角度来看待：由于 IDP 区域能够实现瞬态结构排列，因此每个构象体在细胞内都可以发挥不同的作用。然而，由于 IDP 区域很难通过经典技术（针对具有独特构象的球状蛋白质进行优化）进行研究，因此需要新的方法。在这里，我们使用登革热病毒（DENV）衣壳（C）蛋白和链球菌蛋白 G 的免疫球蛋白结合域，描述了一种简单的 NMR 方法，可区分结构域和 IDP 区域中单氨基酸 N-H 基团的溶剂可及性。我们还深入了解了 DENV C 灵活折叠区域的生物学活性。该方法基于最小的 pH 变化，使用成熟的 H-N HSQC 脉冲序列，并且易于在当前的蛋白质 NMR 常规中实现。生成的数据易于解释，这种快速方法是一种有用的 IDPs 特征描述方法的首选。
