Core D2: Chemical Synthesis

化学核心关注的是合成化学的应用，以阐明作为桥接和试点项目中概述的研究调查主题的膜蛋白类的分子功能基础。该核心将提供最先进的技术，将生物物理探针引入到广泛的分子实体中，包括表达蛋白质、半合成蛋白质、嵌合蛋白质和肽，用于体外研究和自然细胞环境中的体内研究。合成方法的力量在于，我们可以设计和合成具有新特性的蛋白质分子，这些蛋白质分子可以根据所提出的特定生物学问题以及用于解决这些问题的生物物理工具进行微调。现有的方法，包括全蛋白合成， 表达的蛋白质连接、化学标签和非天然氨基酸诱变可用于在桥接项目中立即实施。此外，我们寻求开拓下一代这些技术的开发和实施，这些技术包括光谱探针，将提供 以前所未有的多功能性和精确度进行分子运动。我们将与计算核心和蛋白质生产核心密切合作，利用化学方法将这些探针战略性地定位在表达的膜蛋白或合成蛋白中，以提供功能，构象和动态变化的最翔实的图片。 我们的努力是出于迫切需要设计几类下一代光谱探针，以精确剖析膜蛋白功能中构象变化的作用。我们的总和半合成的一般方法的目的是标记蛋白质在一个非或最小干扰的方式与探针核作为当地的报告基团。这将极大地扩大探索的可能性，迄今为止，无法实现的功能蛋白质动力学使用高分辨率核磁共振（NMR）和傅立叶变换红外（FTIR）光谱。此外，化学蛋白质合成和半合成还能够将荧光和EPR标记精确地、位点特异性地引入复杂的蛋白质系统中，以将动态特性与生物功能相关联。 将与桥接项目一起开发具有高分辨率（例如单分子和超分辨率）成像专用特性的下一代化学标签探针，以及使用misacylated tRNA在体内选择性标记单个蛋白质的策略，以提供所需的工具膜蛋白动态的分子解析，即使在活细胞中也是如此。

The Chemistry Core is concerned with the application of synthetic chemistry to the elucidation of the molecular basis of function for the classes of integral membrane proteins that are the subjects of the research investigations outlined in the Bridging and Pilot projects. The Core will provide state-of-the-art technology to introduce biophysical probes into a wide range of molecular entities including expressed proteins, semisynthetic proteins, chimeric proteins and peptides, for in vitro studies and also for in vivo studies in the natural cellular environment. The power of the synthetic approach is that we can design and synthesize protein molecules with novel properties that are finely tuned to the specific biological questions being asked and to the biophysical tools that will be used to address them. Existing methods, including total protein synthesis, expressed protein ligation, chemical tags, and unnatural amino acid mutagenesis are available for immediate implementation in the bridging projects. Moreover, we seek to pioneer the development and implementation of the next generation of these technologies incorporating spectroscopy probes that will provide for readouts of molecular motions with unprecedented versatility and precision. We will work closely with both the Computational Core and the Protein Production Core to use chemistry to strategically position these probes within expressed integral membrane proteins or synthetic proteins to provide the most informative picture of functional, conformational, and dynamic changes. Our efforts are motivated by a compelling need to design several classes of next generation spectroscopic probes to precisely dissect the role of conformational changes in membrane protein function. Our general methods for total and semi-synthesis are designed to label proteins in a non- or minimally-perturbing fashion with probe nuclei to act as local reporter groups. This will greatly expand the possibilities to explore, heretofore, unattainable features of protein dynamics using high-resolution nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopy. Additionally, chemical protein synthesis and semi-synthesis also enables the precise, site-specific introduction of fluorescent and EPR labels into complex protein systems to correlate dynamic properties with biological function. Next generation chemical tag probes with specialized properties for high-resolution (e.g. single molecule and super-resolution) imaging, and strategies for selectively labeling individual proteins in vivo using misacylated tRNAs, will be developed with the bridging projects in order to provide the tools needed for molecular resolution of membrane protein dynamics, even in living cells.