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首页> 外文期刊>Journal of Molecular Biology >Selection and structure of hyperactive inteins: peripheral changes relayed to the catalytic center.
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Selection and structure of hyperactive inteins: peripheral changes relayed to the catalytic center.

机译：高活性蛋白的选择和结构：外围变化传递至催化中心。

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摘要

Inteins are phylogenetically diverse self-splicing proteins that are of great functional, evolutionary, biotechnological, and medical interest. To address the relationship between intein structure and function, particularly with respect to regulating the splicing reaction, and to groom inteins for application, we developed a phage display system to extend current in vivo selection for enhanced intein function to selection in vitro. We thereby isolated inteins that can function under excursions in temperature, pH, and denaturing environment. Remarkably, most mutations mapped to the surface of the intein, remote from the active site. We chose two mutants with enhanced splicing activity for crystallography, one of which was also subjected to NMR analysis. These studies define a "ripple effect", whereby mutations in peripheral non-catalytic residues can cause subtle allosteric changes in the active-site environment in a way that facilitates intein activity. Altered salt-bridge formation and chemical shift changes of the mutant inteins provide a molecular rationale for their phenotypes. These fundamental insights will advance the utility of inteins in chemical biology, biotechnology, and medicine.
机译：内含子是系统发育多样的自剪接蛋白，具有重要的功能，进化，生物技术和医学意义。为了解决intein结构与功能之间的关系，特别是在调节剪接反应方面，以及修饰intein的应用，我们开发了一种噬菌体展示系统，以扩展当前体内选择的范围，以增强intein的功能至体外选择。因此，我们分离出了可以在温度，pH和变性环境漂移下起作用的内含子。值得注意的是，大多数突变都映射到内含蛋白表面，远离活性位点。我们选择了两个具有增强的剪接活性的突变体用于晶体学，其中一个也进行了NMR分析。这些研究定义了“涟漪效应”，即外围非催化残基中的突变可以以促进内含肽活性的方式在活性位点环境中引起细微的变构变化。突变的intein改变的盐桥形成和化学位移变化为它们的表型提供了分子原理。这些基本见解将促进内含肽在化学生物学，生物技术和医学中的应用。

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