INTEIN MECHANISMS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 一项研究计划正在进行中，以研究内含肽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过程，其中一个插入序列，称为内含肽，从宿主蛋白质，外显肽去除。在蛋白质反式剪接中，内含肽被分成两部分，并且剪接仅在这些片段重构时发生。内含肽存在于所有3个系统发育域的单细胞生物体中，包括几种病原体。此外，所有多细胞生物体都含有在成熟过程中经历自蛋白水解反应的蛋白质，这些蛋白质可能以类似于内含肽的方式催化分子内肽键的裂解。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，但我们对内含肽如何催化和调节这些步骤的知识还不太发达。因此，有必要研究这一过程的详细机制。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，而且对剪接抑制剂的设计和蛋白质剪接实际应用的进一步发展也至关重要。我们有一系列的假设，有关内含肽如何协调级联的化学步骤，他们催化和反式剪接内含肽如何相互作用和折叠效率高。因此，我们已经制备了几个内含肽类似物含有非天然氨基酸，同位素探针和异肽连接，然后采用这些在动力学，热力学和结构的研究蛋白质剪接在顺式和反式。我们希望使用标准方法模拟剪接反应，并将结果与实验观察结果进行比较。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A research program is underway 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 likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. 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. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. We have a series of hypotheses related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we have prepared several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employed these in kinetic, thermodynamic and structural investigations of protein splicing in cis and in trans . We wish to simulate the splicing reactions using standard methods and compare the results to experimental observation .