RUI: Defining the thermodynamics of MarR family transcription factor interactions with DNA

MARR家族蛋白存在于大多数细菌物种中。这些蛋白质与DNA结合非常紧密，并调节基因的表达，使细菌能够对各种形式的环境压力做出反应，包括有毒化学品和抗生素。MARR蛋白用一个共同的结构基序与DNA结合，但每个蛋白质都针对一个特定的序列。这些蛋白质与其目标DNA序列的亲和力的能量基础还不是很清楚。该项目旨在确定推动与DNA关联的Marr蛋白的结构成分。这些研究将提供对MarR蛋白质如何与DNA相互作用的详细了解，并有望从总体上更深入地了解序列特异性蛋白质-DNA相互作用的能量学。此外，这些研究将首次描述MarR蛋白在调节致病细菌艰难梭菌基因中的作用，并提供对这种生物对不断变化的环境条件和压力的反应，包括对抗生素和抗菌剂的耐药性的洞察。该项目将在一个以本科生为主的机构进行，并为将参加该项目所有阶段的本科生提供实质性培训机会。这群不同的学生将利用生物化学的现代技术进行独立研究，这将为他们在STEM劳动力中的深造和职业生涯做好准备。MarR蛋白通过一个有翼的螺旋转弯螺旋(WHTH)基序与DNA结合，几乎所有的相互作用都涉及wHTH的“识别螺旋”和“翼”，它们分别与DNA的主槽和副槽形成碱基特异的接触。然而，人们对这些高亲和力缔合的热力学知之甚少。虽然DNA结合蛋白传统上被归类为主要的沟槽结合蛋白或次要的沟槽结合蛋白，每一类都与不同的热力学特征相关联，但MarR蛋白不能巧妙地归入这两类蛋白质中的任何一类，因为它们通过两个沟槽中广泛的碱基特定接触与DNA结合。该项目的第一个目标是确定是否存在表征Marr家族与DNA结合作用的热力学特征，并分析蛋白质与主要和次要凹槽相互作用对结合的能量贡献。更广泛地说，这些发现将检验流行的观点，即这种相互作用每一个都有一个独特的热力学特征。这些目标将通过对涉及六个MARR同源物的DNA结合热力学的系统分析来实现，其中包括两个先前在结构上定义的MARR同源物，以及来自艰难梭菌的四个以前未确定特征的MARR同源物。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

MarR family proteins are found in most bacterial species. These proteins bind very tightly to DNA and regulate the expression of genes that allow the bacterium to respond to various forms of environmental stress, including noxious chemicals and antibiotics. MarR proteins bind DNA with a common structural motif, yet each is specific for a particular sequence. The energetic basis for the affinity of these proteins for their target DNA sequences is not well understand. This project seeks to determine the structural components of MarR proteins that drive association with DNA. These studies will provide a detailed understanding of how MarR proteins interact with DNA, and are also expected to provide deeper insight into the energetics of sequence-specific protein-DNA interactions in general. In addition, these investigations will provide the first description of the roles of MarR proteins in regulating genes in the pathogenic bacterium Clostridium difficile and offer insight into this organism’s response to changing environmental conditions and stresses, including its resistance to antibiotics and antimicrobials. The project will be conducted at a primarily undergraduate institution and provide substantive training opportunities for undergraduate students who will participate in all phases of the project. This diverse group of students will conduct independent research using modern techniques in biochemistry, which will prepare them for advanced studies and careers in the STEM workforce. MarR proteins associate with DNA through a winged-helix-turn-helix (wHTH) motif, with nearly all interactions involving the wHTH “recognition helix” and “wing” which form base-specific contacts with the DNA major and minor grooves, respectively. However, the thermodynamics of these high-affinity associations is poorly understood. While DNA binding proteins have traditionally been classified as either major groove binders or minor groove binders, with each class being associated with a distinct thermodynamic signature, MarR proteins do not fall neatly into either category since they associate with DNA through extensive base-specific contacts in both grooves. The first objective of this project is to determine if there is a thermodynamic signature that characterizes MarR family binding interactions with DNA and to parse the energetic contributions to binding that stems from protein interactions with the major and minor grooves. More broadly, these findings will test the prevailing view that such interactions each have a characteristic thermodynamic signature. These objectives will be met through a systematic analysis of DNA binding thermodynamics involving six MarR homologs, including two which were previously structurally-defined, as well as four previously uncharacterized MarR homologs from the bacterium, Clostridium difficile.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.