Segmental Labeling for Protein Structure by NMR

通过 NMR 对蛋白质结构进行分段标记

• 批准号: 6777484
• 负责人: Tom Muir
• 金额: $ 43.42万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6777484
• 关键词: bioimaging /biomedical imaging; esterification; nuclear magnetic resonance spectroscopy; protein structure; recombinant proteins; stable isotope double label; structural biology; technology /technique development; thioester

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.

描述（由申请人提供）：将进行一项研究计划，以研究Inteins如何催化和调节蛋白质剪接和蛋白质反式剪接的各种步骤。蛋白质剪接是一种翻译后过程，其中将内部序列的中间序列从宿主蛋白（外te）中取出。在蛋白质移植中，内部内部的内部分为两块，仅在重构这些片段后才发生剪接。来自所有3个系统发育结构域（包括几种病原体）的单细胞生物中存在于网状素。此外，所有多细胞生物都包含在成熟过程中经历自蛋白解反应的蛋白质，并且可能以类似于输入的方式催化肽键的分子内切割。因此，了解油素如何催化蛋白质剪接中的步骤通常可以作为理解自动蛋白解体机制的范例。虽然我们对蛋白质剪接的基本化学步骤有合理的了解，但我们对Inteins如何催化和调节这些步骤的了解较少。我们将通过制备含有不自然氨基酸和稳定同位素探针的半合成肠内分子来解决这种情况。该研究计划的关键是蛋白质半合成技术表达的蛋白质连接，它允许将非天然氨基酸和同位素探针特定于现场引入大型蛋白质。这些节段标记的分子的可用性将使我们能够进行一系列同位素编辑的NMR光谱研究，这些研究有望揭示Inteins如何催化和调节CIS和Trans中的蛋白质剪接过程。这些信息不仅将加深我们对蛋白质剪接和相关过程的理解，而且还将对蛋白质剪接的实际应用进一步开发。因此，在程序的最后一部分中，我们将优化并扩展我们最近引入的方法，条件蛋白质移媒体（CPS），该方法允许蛋白质剪接由小分子雷帕霉素触发。具体而言，我们将研究CP是否可以通过开发用于蛋白激酶的一般开关来控制蛋白质功能。然后，我们将探讨该方法在活细胞中是否起作用以及在这种情况下的范围和局限性。我们预计这些研究将导致开发用于控制蛋白质结构和体内功能的通用车辆，并具有许多生物学应用。