Allosteric Regulation of the Nickel-dependent NikR Repressor

智力上的优点。 过渡金属是所有活细胞的必需营养素，但过量它们是有毒的。生物利用几种机制来确保金属水平得到严格控制。微生物通常使用金属敏感DNA结合蛋白来控制金属转运蛋白的转录调节，从而调节响应于细胞内金属水平的金属转运量。 该项目的重点是镍依赖性NikR转录调节因子，这是一种在各种细菌和古细菌物种中发现的蛋白质。该蛋白包含两个不同的结构模块：N-末端DNA结合结构域和C-末端镍结合结构域。NikR仅在镍存在的情况下结合DNA，因此用于检测细胞内过量的镍。该项目旨在了解镍如何改变NikR结构以实现DNA结合。使用计算和实验方法的组合，镍和DNA结合结构域之间的变构通信所需的氨基酸残基将被确定。计算方法将表征在镍存在下改变的相互作用网络和大尺度运动。诱变方法将用于鉴定对NikR的镍依赖性反应至关重要的氨基酸。这些实验旨在破坏这些相互作用，或者利用遗传方法的力量，识别绕过DNA结合所需的镍的突变体。这些残基的结构映射提供了与计算方法的接触点。更广泛的影响。该项目的跨学科性质通过提供更广泛的实验方法对学生的培训产生直接影响。该项目的实验目标非常适合早期的研究经验，暴露在微生物生理学和遗传学，蛋白质结构和功能，以及生物无机化学的基本概念。这项研究的参与者包括学生和教师作为研究科学家（星）暑期项目和华盛顿大学青年科学家计划（YSP）的学生，这两个项目都为来自不同背景的圣路易斯地区高中生提供暑期研究经验。

Intellectual merit. Transition metals are an essential nutrient for all living cells, yet they are toxic in excess amounts. Organisms utilize several mechanisms to ensure metal levels are carefully controlled. Microbes commonly use metal-sensing DNA-binding proteins to control transcriptional regulation of metal transporters, thereby regulating the amount of metal transport in response to intracellular metal levels. This project focuses on the nickel-dependent NikR transcriptional regulator, a protein found in a wide variety of bacterial and archaeal species. The protein contains two distinct structural modules: an N-terminal DNA-binding domain and a C-terminal nickel binding domain. NikR binds DNA only in the presence of nickel and thus serves to sense excess nickel inside the cell. This project seeks to understand how nickel alters the NikR structure to enable DNA binding. Using a combination of computational and experimental approaches, the amino acid residues required for allosteric communication between the nickel- and DNA-binding domains will be identified. Computational methods will characterize interaction networks and large scale motions that are altered in the presence of nickel. Mutagenesis approaches will be used to identify amino acids that are critical for the nickel-dependent response of NikR. The experiments are designed to either disrupt these interactions or, using the power of genetic approaches, identify mutants that bypass the nickel requirement for DNA-binding. Structural mapping of these residues provides a point of contact with computational methods. Broader impacts. The interdisciplinary nature of this project has a direct impact on the training of students by providing access to a wider variety of experimental methodologies. The experimental objectives of the project are well suited to early research experiences by exposing to fundamental concepts in microbial physiology and genetics, protein structure and function, and bioinorganic chemistry. Participants in this research include students from the Students and Teachers as Research Scientists (STARS) summer program and the Washington University Young Scientist Program (YSP) both of which provide summer research experiences for St. Louis area high school students from diverse backgrounds.