Clark Evan T, Sievers Elanor E, Debelouchina Galia T
Department of Chemistry and Biochemistry, Division of Physical Sciences, University of California, San Diego, La Jolla, CA 92093, U.S.A.
Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, U.S.A.
J Magn Reson Open. 2022 Jun;10-11. doi: 10.1016/j.jmro.2022.100044. Epub 2022 Feb 18.
Among structural biology techniques, NMR spectroscopy offers unique capabilities that enable the atomic resolution studies of dynamic and heterogeneous biological systems under physiological and native conditions. Complex biological systems, however, often challenge NMR spectroscopists with their low sensitivity, crowded spectra or large linewidths that reflect their intricate interaction patterns and dynamics. While some of these challenges can be overcome with the development of new spectroscopic approaches, chemical biology can also offer elegant and efficient solutions at the sample preparation stage. In this tutorial, we aim to present several chemical biology tools that enable the preparation of selectively and segmentally labeled protein samples, as well as the introduction of site-specific spectroscopic probes and post-translational modifications. The four tools covered here, namely cysteine chemistry, inteins, native chemical ligation, and unnatural amino acid incorporation, have been developed and optimized in recent years to be more efficient and applicable to a wider range of proteins than ever before. We briefly introduce each tool, describe its advantages and disadvantages in the context of NMR experiments, and offer practical advice for sample preparation and analysis. We hope that this tutorial will introduce beginning researchers in the field to the possibilities chemical biology can offer to NMR spectroscopists, and that it will inspire new and exciting applications in the quest to understand protein function in health and disease.
在结构生物学技术中，核磁共振光谱提供了独特的能力，能够在生理和天然条件下对动态和异质生物系统进行原子分辨率研究。然而，复杂的生物系统常常给核磁共振光谱学家带来挑战，它们灵敏度低、光谱拥挤或线宽较大，反映出其复杂的相互作用模式和动力学。虽然其中一些挑战可以通过开发新的光谱方法来克服，但化学生物学也可以在样品制备阶段提供巧妙而有效的解决方案。在本教程中，我们旨在介绍几种化学生物学工具，这些工具能够制备选择性和分段标记的蛋白质样品，以及引入位点特异性光谱探针和翻译后修饰。这里介绍的四种工具，即半胱氨酸化学、内含肽、天然化学连接和非天然氨基酸掺入，近年来得到了开发和优化，比以往任何时候都更高效，适用于更广泛的蛋白质。我们简要介绍每种工具，描述其在核磁共振实验中的优缺点，并提供样品制备和分析的实用建议。我们希望本教程能让该领域的新手研究人员了解化学生物学为核磁共振光谱学家提供的可能性，并激发人们在探索健康和疾病中蛋白质功能的过程中开展新的、令人兴奋的应用。
