Segmental Labeling for Protein Structure by NMR

项目描述（由申请人提供）：研究蛋白如何催化和调节蛋白质剪接和蛋白质反式剪接的各个步骤。蛋白质剪接是一个翻译后的过程，在这个过程中，一个被称为内蛋白的中间序列从宿主蛋白质（外蛋白）上被移除。在蛋白质反式剪接中，内部蛋白被分成两个片段，剪接仅在这些片段重组时发生。蛋白存在于所有3个系统发育域的单细胞生物中，包括几种病原体。此外，所有多细胞生物都含有在成熟过程中进行自身蛋白水解反应的蛋白质，这些蛋白质可能以类似于蛋白质的方式催化肽键的分子内裂解。因此，了解内部蛋白如何催化蛋白质剪接的步骤可以作为理解自身蛋白水解机制的范例。虽然我们对蛋白质剪接的基本化学步骤有了一个合理的了解，但我们对蛋白质如何催化和调节这些步骤的了解还不太完善。我们将通过制备含有非天然氨基酸和稳定同位素探针的半合成蛋白分子来解决这一问题。这部分研究计划的关键是蛋白质半合成技术，表达蛋白连接，它允许非天然氨基酸和同位素探针被位点特异性地引入到大蛋白质中。这些片段标记分子的可用性将使我们能够进行一系列同位素编辑核磁共振波谱研究，这些研究有望揭示内部蛋白是如何催化和调节顺式和反式蛋白质剪接过程的。这些信息不仅加深了我们对蛋白质剪接及其相关过程的理解，而且对进一步开发蛋白质剪接的实际应用也很有帮助。因此，在项目的最后一部分，我们将优化和扩展我们最近引入的方法，条件蛋白质反式剪接（CPS），该方法允许由小分子雷帕霉素触发蛋白质剪接。具体来说，我们将研究CPS是否可以通过开发蛋白质激酶的通用开关来控制蛋白质功能。然后，我们将探讨这种方法是否适用于活细胞，以及在这种情况下的范围和局限性是什么。我们预计这些研究将导致一种控制体内蛋白质结构和功能的通用载体的发展，并将有许多生物学应用。

DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.