Allosteric Regulation of the Nickel-dependent NikR Repressor

智力上的优点。过渡金属是所有活细胞必不可少的营养物质，但过量的过渡金属是有毒的。生物体利用几种机制来确保金属水平得到仔细控制。微生物通常使用金属感应DNA结合蛋白来控制金属转运体的转录调控，从而调节金属转运体的量以响应细胞内的金属水平。这个项目的重点是镍依赖的NikR转录调节因子，这是一种在各种细菌和古生菌物种中发现的蛋白质。该蛋白含有两个不同的结构模块：N端DNA结合域和C端镍结合域。NikR只有在镍存在的情况下才能与DNA结合，因此可以感觉到细胞内多余的镍。这个项目试图了解镍是如何改变NikR结构以实现DNA结合的。使用计算和实验相结合的方法，将确定镍和DNA结合结构域之间变构通信所需的氨基酸残基。计算方法将表征相互作用网络和大规模运动，这些运动在镍的存在下发生变化。突变方法将被用来识别对NikR的镍依赖反应至关重要的氨基酸。这些实验的目的是要么破坏这些相互作用，要么利用遗传方法的力量，识别绕过DNA结合所需镍的突变。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对学生的培训产生了直接影响，因为它提供了更广泛的实验方法。该项目的实验目标通过暴露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，非常适合早期研究经验。这项研究的参与者包括学生和教师作为研究科学家(STAR)暑期计划和华盛顿大学青年科学家计划(YSP)的学生，这两个计划都为来自不同背景的圣路易斯地区的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.