NMR Methods to decipher the structural and dynamics aspects of TCR mechanobiology

摘要 为了保护我们免受无数疾病的侵害，适应性免疫需要T细胞识别蛋白质衍生肽 在异常细胞表面表达的外源、突变或其他异常来源的。T细胞中枢 功能是细胞表面活化的T细胞受体（TCR），其识别这些结合到主要抗原的各种肽。 组织相容性分子（pMHC）。虽然对TCR的静态构象有很多了解， 分子和它们的pMHC配体以及所得的连接复合物，仍然不知道确切是什么。 发生在TCR-pMHC复合物中，产生不同的信号传导结果， 应答最近的实验已经强调了TCR-pMHC连接和信号传导的动态性质， 产生T细胞反应所需的关键力量输入。这意味着一个动态的系统， 输入皮牛顿（pN）量的力以产生信号准备TCR蛋白。我们建议 开发NMR方法，用于研究TCR机械生物学中涉及的大细胞外结构域，包括 TCR及其发育前体preTCR以及处于NMR极限的pMHC配体 观察.这包括不能在细菌系统中表达的蛋白质结构域， 容易被全氘代，目前的标准标记策略，解决高分子量蛋白质 系统.因此，在目标1中，我们采用直接15 N-检测方法，其不需要蛋白质全氘代 用于骨架共振分配。此外，我们将开发新的13 C检测实验与TROSY 增强产生高分辨的芳香侧链光谱。为了提高核磁共振技术的水平 在上面的技术中，在目标2中，我们将建立新的标记方案来帮助破译结构， preTCR、TCR和pMHC的动力学。为了解决这些大蛋白质的共振分配问题，我们将继续研究 使用“混合丙酮酸盐”作为碳源来标记蛋白质以获得残基特异性模式。串联 目的1是通过化学酶法制备13 C和13 C-19 F标记的芳香族氨基酸 合成.由于我们打算研究的TCR系统的一些蛋白质组分不能重组， 在大肠杆菌中表达，我们将追求在真核系统中的表达，其中完全氘代是一个挑战。 此处开发的标签技术将转移至核心B。在目标3中，我们将使用NMR技术 和标记方法，以获得关于TCR-pMHC的结构和动力学的信息 配合物特别是弛豫色散和CEST，我们将利用19 F核作为探针来访问动态 在低微秒的时间尺度上。提取的动态信息将与MD核心C一起使用， 通过计算机观察动态，并将NMR中测量的动态和构象状态与 在武力下观察。原子发现的功能影响将在项目1和2中评估， 突变迭代这里锻造的核磁共振方法将照亮众所周知的机械盲点 对TCR激活的理解，已经在目标3中通过激发初步隐藏状态数据进行了预览。

ABSTRACT To protect us from myriad diseases, adaptive immunity requires T cell recognition of protein-derived peptides of foreign, mutant, or otherwise anomalous origin expressed on the surface of aberrant cells. Central to T cell function is the cell surface  T cell receptor (TCR), which recognizes these various peptides bound to major histocompatibility molecules (pMHC). While a great deal is known about the static conformations of TCR molecules and their pMHC ligands as well as the resultant ligation complexes, it is still unknown precisely what happens within the TCR-pMHC complex to generate the diverse signaling outcomes which drive T cell responses. Recent experiments have highlighted the dynamic nature of TCR-pMHC ligation and signaling, with a critical input of force necessary to generate T cell responses. This implies a dynamic system, poised to signal with the input of piconewton (pN) amounts of force to generate signaling-ready TCR proteins. We propose to develop NMR methods for studies of large extracellular domains involved in TCR mechanobiology, including the TCR and its developmental precursor, the preTCR, as well as the pMHC ligands that are at the limit of NMR observation. This includes protein domains that cannot be expressed in bacterial systems and thus cannot readily be perdeuterated, a current standard labeling strategy for addressing high molecular weight protein systems. Thus, in Aim 1 we employ direct 15N-detection methods, which do not require protein perdeuteration for backbone resonance assignment. Further, we will develop new 13C-detected experiments with TROSY enhancement that yield highly resolved spectra of aromatic side chains. To augment the state of the art NMR technology above, in Aim 2 we will establish new labeling schemes to aid in deciphering the structure and dynamics of preTCR, TCR and pMHC. To tackle the resonance assignment of these large proteins we will pursue the use of “mixed pyruvate” as a carbon source to label proteins to obtain residue specific patterns. In tandem with Aim 1 we will produce isolated 13C and 13C-19F labeled aromatic amino acids through chemoenzymatic synthesis. Since some protein components of the TCR systems we intend to study cannot be recombinantly expressed in E.Coli, we will pursue expression in eukaryotic systems, where complete deuteration is a challenge. The labeling technology developed here will be transferred to Core B. In Aim 3, we will use the NMR technologies and labeling methods, described above, to obtain information about structure and dynamics of TCR-pMHC complexes. In particular relaxation dispersion and CEST, we will leverage 19F nuclei as probe to access dynamics in the low microsecond time scale. The extracted dynamics information will be utilized with the MD Core C to observe dynamics in silico and link the dynamics and conformational states measured in NMR to those that are observed under force. Functional impact of the atomistic findings will be assessed in Projects 1 and 2 through mutational iterations. The NMR methods forged here will illuminate the proverbial blind spot in mechanistic understanding of TCR activation, already previewed via exciting preliminary hidden state data in Aim 3.