Structural investigation of an immunoglobulin domain: biosynthesis on the ribosome and co-translational folding

蛋白质折叠是多肽获得其正确的三维生物活性功能的过程。在核糖体的生物合成过程中，伸长的新生链以载体的方式从核糖体出口通道的受限环境中出现，并开始探索构象空间。对新出现的新生链的结构理解是稀疏的，因为其固有的动力学使得传统的结构和生物物理方法（包括传统的核磁共振）无法看到它。顺磁弛豫增强（PRE）核磁共振的应用不仅有助于主要填充态的结构表征，而且有助于检测和表征共平移折叠固有的快速弛豫次要态。PRE方法可探测距离达15-24 Å的总体平均瞬态接触。如前所述，对于分离的蛋白质，氮氧化物自旋标签附着在蛋白质的一个区域上，并且在其氧化（顺磁）状态下，增强异核相干的弛豫。松弛增强尺度为r-6，具有标签接近性，允许计算用于通过MD模拟计算结构集成的距离约束。使用这种方法，我将从结构上研究共翻译折叠的内在动态过程，使用免疫球蛋白结构域作为我的模型蛋白，并对这个迄今为止结构上看不见的过程有更深入的了解。

Protein folding is the process by which a polypeptide acquires its correct threedimensional, biologically active function. During biosynthesis on the ribosome the elongating nascent chain emerges in a vectorial manner from the restrictedenvironment of the ribosomal exit tunnel and begins to explore conformational space. The structural understanding of the emerging nascent chain is sparse because its inherent dynamics is making it invisible to conventional structural and biophysical methods, including conventional NMR. Application of paramagnetic relaxation enhancement (PRE) NMR cannot only aid in structural characterization of the major populated state but can also aid in detecting and characterizing fast relaxing minor states which are inherent to co-translational folding.The PRE method probes ensemble-averaged, transient contacts over distances as great as 15-24 Å. As previously described for isolated proteins, a nitroxide spin-label is attached to a region of a protein, and in its oxidized (paramagnetic) state, enhances relaxation of heteronuclear coherences. Relaxation enhancement scales as r-6 with label proximity, allowing the computation of distance restraints used to calculate ensembles of structures by MD simulations.Using this approach, I will structurally investigate the inherently dynamic process of cotranslational folding using an immunoglobulin domain as my model protein and get a deeper understanding of this so far structurally invisible process.