Segmental Labeling for Protein Structure by NMR

描述(由申请人提供)：将开展一项研究计划，研究内含子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，一个被称为内含子的中间序列被从宿主蛋白质外显子上移除。在蛋白质的反式剪接中，内含子被分成两个片段，只有在这些片段重组时才会发生剪接。内含子存在于所有3个系统发育域的单细胞生物体中，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发生自蛋白分解反应的蛋白质，这些蛋白质可能以类似于内含子的方式催化多肽键的分子内断裂。因此，了解内含子是如何催化蛋白质剪接中的步骤的，可能会成为一般理解自蛋白降解机制的范例。虽然我们对蛋白质剪接中的基本化学步骤有了合理的了解，但我们对内含子如何催化和调节这些步骤的了解还不够充分。我们将通过制备含有非天然氨基酸的半合成内含素分子和稳定的同位素探针来解决这一问题。这部分研究计划的关键是蛋白质半合成技术，即表达蛋白质连接，该技术允许将非天然氨基酸和同位素探针定位地引入大型蛋白质中。这些片段标记分子的可获得性将使我们能够进行一系列同位素编辑的核磁共振波谱研究，有望揭示内含子如何催化和调节顺式和反式的蛋白质剪接过程。这些信息不仅将加深我们对蛋白质剪接及其相关过程的理解，也将有助于进一步发展蛋白质剪接的实际应用。因此，在程序的最后部分，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)，该方法允许小分子雷帕霉素触发蛋白质剪接。具体地说，我们将研究CPS是否可以通过开发一种通用的蛋白激酶开关来控制蛋白质的功能。然后，我们将探索这种方法是否适用于活细胞，以及在这方面的范围和限制是什么。我们预计，这些研究将导致开发一种在体内控制蛋白质结构和功能的通用工具，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.