REDOX-SENSING REPRESSOR

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 分子氧是有氧生命的基本要求，但氧化应激会损害大分子，如果不加以控制可能是致命的。到目前为止，调节转录以响应氧化还原状态的变构构象变化很少，如果有的话。这项拟议研究的长期目标是阐明调节DNA结合的新的和基本的变构机制，以响应细胞内的氧化还原状态。 在革兰氏阳性细菌中，氧化还原敏感抑制因子(REX)采用了一种巧妙的方法来区分基本辅因子NAD的还原和氧化状态。在氧化的NAD+存在的情况下，REX结合DNA靶标并抑制呼吸基因的转录，但当还原的NADH水平异常上升时，REX授权转录。该实验室先前确定了Rex/NADH复合体的X射线结构。虽然Rex/NADH结构提供了一个无法识别DNA的Rex状态的重要视图，但Rex对NADH/NAD+的变构反应的基本机制仍不清楚。需要了解Rex/NAD+/DNA复合体的结构才能了解Rex如何重新排列以识别DNA，以及NAD+/NADH之间的微小差异如何触发这种转变。 为了实现这一目标，人们已经获得了Rex/NAD+/DNA共晶体。从溶解晶体的A280：A260比率中证实了DNA和蛋白质的存在。虽然晶体的衍射率超过3？分辨率，但300？c轴限制了我们在内部收集完整、高分辨率数据的能力。结构将通过用溴化DNA进行MIR或MAD相变来解决，并由每个单体的单个SeMet提供额外的相信息。基于Rex/NADH和DNA的不相容形状，Rex很可能在NADH/NAD+交换后发生较大的构象变化。Rex的假想构象重排将揭示一种新的转录调控范式，以响应NAD+和NADH之间的最小差异。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Molecular oxygen is a fundamental requirement for aerobic life, yet oxidative stress damages macromolecules and can be lethal if unchecked. Thus far, allosteric conformational changes that regulate transcription in response to the redox state have been visualized in few, if any cases. The long term goal of the proposed research is to elucidate novel and essential allosteric mechanisms that regulate DNA-binding in response to the intracellular redox state. In Gram-positive bacteria, the redox-sensing repressor (Rex) adopts the elegant approach of distinguishing the reduced and oxidized states of the essential cofactor, NAD. Rex binds the DNA target and represses transcription of respiratory genes in the presence of oxidized NAD+, yet authorizes transcription when reduced NADH levels abnormally rise. This laboratory previously determined the X-ray structure of the Rex/NADH complex. Although the Rex/NADH structure provided one important view of the state of Rex that is unable to recognize DNA, the fundamental mechanism of the allosteric response of Rex to NADH/NAD+ remains unknown. The structure of the Rex/NAD+/DNA complex is needed to understand how Rex rearranges to recognize DNA, and how the slight differences between NAD+/NADH trigger this transition. Towards this goal, Rex/NAD+/DNA cocrystals have been obtained. The presence of both DNA and protein was verified from the A280:A260 ratio of dissolved crystals. Although the crystals diffract beyond 3¿¿¿ resolution, a 300¿¿¿ c-axis limits our ability to collect complete, high-resolution data in-house. The structure will be solved by MIR or MAD phasing with brominated-DNA, and additional phase information provided by a single SeMet per monomer. Based on the incompatible shapes of Rex/NADH and DNA, Rex is likely to undergo a large conformational change following NADH/NAD+ exchange. The hypothesized conformational rearrangement of Rex would reveal a new paradigm for transcriptional regulation in response to the minimal differences between NAD+ and NADH.