Core D2: Chemical Synthesis

化学核心涉及应用合成化学来阐明作为桥接和试点项目中概述的研究调查的主题的整膜蛋白类别的功能的分子基础。该核心将提供最先进的技术，将生物物理探测器引入广泛的分子实体，包括表达蛋白质、半合成蛋白质、嵌合蛋白质和多肽，用于体外研究，也用于在自然细胞环境中的体内研究。合成方法的强大之处在于，我们可以设计和合成具有新性质的蛋白质分子，这些分子可以精确地调整到所提出的特定生物学问题和将用于解决这些问题的生物物理工具。现有的方法，包括总蛋白质合成， 表达的蛋白质连接、化学标签和非天然氨基酸突变可在桥梁项目中立即实施。此外，我们寻求率先开发和实施下一代这些技术，包括光谱探头，将提供读数 分子运动具有前所未有的多功能性和精确度。我们将与计算核心和蛋白质生产核心密切合作，利用化学手段在表达的整膜蛋白质或合成蛋白质中战略性地定位这些探针，以提供功能、构象和动态变化的最具信息量的图像。 我们努力的动机是迫切需要设计几类下一代光谱探测器，以精确剖析膜蛋白功能构象变化的作用。我们一般的全合成和半合成方法是用探针核作为局部报告基团，以无扰动或最小扰动的方式标记蛋白质。这将极大地扩大利用高分辨率核磁共振(核磁共振)和傅里叶变换红外(FTIR)光谱探索迄今无法获得的蛋白质动力学特征的可能性。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、特定地引入复杂的蛋白质系统，以将动态特性与生物功能相关联。 下一代具有高分辨率(如单分子和超分辨率)成像特性的化学标记探针，以及使用错酰化tRNA选择性标记体内单个蛋白质的策略，将与桥梁项目一起开发，以便提供即使在活细胞中也是膜蛋白质动力学的分子解析所需的工具。

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.