Nmr Studies Of Biomolecular Structure, Function, And Dynamics

独立项目：叶酸依赖性酶在单碳代谢和核苷酸生物合成中起核心作用;其扰动可能是致突变的并最终致死。更一般地，叶酸代谢的失调似乎与基因组甲基化、稳定性和修复的缺陷广泛相关。或者，抗叶酸药物如甲氨蝶呤广泛用于治疗肿瘤疾病，但低剂量已发现越来越多地应用于治疗类风湿性疾病和与慢性炎症相关的其他疾病。我们以前的叶酸代谢研究表明，NSAID芬布芬能够与酶二氢叶酸还原酶（DHFR）通过结合到与对氨基苯甲酰基-L-谷氨酸（pABG）相互作用的叶酸结合位点的区域。我们目前正在对其他NSAID与该亚位点的相互作用、它们与该位点结合的能力以及它们对DHFR活性的抑制进行更全面的评估。这些研究已经量化了各种NSAID的DHFR抑制强度，并证明一些具有比芬布芬更大的抑制效力。NSAID的潜在抗叶酸活性提示了在治疗慢性炎性疾病中的额外应用的可能性，而没有更有效的抗叶酸化合物如甲氨蝶呤的一些毒性。 NMR核心活动： 在神经生物学中使用了许多氟化麻醉剂，这些麻醉剂可能难以溶解。 NMR小组利用19 F NMR光谱法评估神经生物学实验室离子通道生理学小组的几种麻醉剂的溶液浓度。 表观遗传学和干细胞生物学实验室的干细胞生物学小组正在研究通过改变DNA碱基的化学性质来产生随机诱变的新方法。 NMR组利用1D NMR评估化学结构和催化反应的有效性，以更好地优化程序。 表观遗传学和干细胞生物学实验室的大分子结构组正在研究RNA结构，以更好地了解结构变化如何影响RNA调控途径。 核磁共振组一直在获取核磁共振数据，以确定RNA靶标的结构。 信号转导实验室的核仁完整性小组正试图了解参与核糖体生物合成途径的分子。 NMR小组获得的数据旨在提供有关las 1/grc 3在生物发生过程中如何与RNA靶点相互作用的信息。 儿科神经内分泌学小组一直对蛋白质SMCHD 1（另一种染色质重塑剂）如何调节ACE 2表达感兴趣，并利用我们的结构生物学计划来确定SMCHD 1是否可能是一个药物靶点。 下调ACE 2表达被认为是治疗SARS-CoV-2感染的潜在方法。 NMR小组已经获得了SMCHD 1构象的数据，并正在帮助为该项目准备样品。 Matrix Biology小组一直对透明质酸（HA）与SARS-CoV-2刺突蛋白的RBD的相互作用感兴趣。 NMR组已经进行了测定以评估不同大小的HA与RBD和刺突蛋白构建体的结合。 如果HA阻断SARS-CoV-2的刺突蛋白与ACE 2的RBD结合，则其可能是有用的治疗剂。 突变和DNA修复调节小组对氧化损伤如何导致突变感兴趣。 该小组目前正在研究各种酵母菌株，以及突变的酵母菌株对氧化化学试剂的反应。 为了更好地了解代谢的影响以及化学途径如何受到氧化化学试剂的影响，NMR小组一直在评估菌株和处理组的酵母代谢组。 评估了代谢途径的相似性和差异性，以与氧化剂的致突变特征进行比较，从而更好地了解致突变的化学机制。 线粒体DNA复制小组试图了解影响细胞器中突变的因素，以维持其自身的基因组。 一个因素可能是由于在线粒体基因组中提出的G四链体附近的UV照射而形成TT二聚体。 NMR组收集化学数据，以基于序列背景和UV剂量评估不同DNA样品中TT二聚体形成的程度。这些数据有助于优化进一步的实验以测量诱变率。 结构细胞生物学小组对DNA损伤如何被识别和修复感兴趣。 在检测DNA损伤中的一种重要蛋白是APTX。 NMR组测量了APTX在各种条件下的反应速率，并通过检查组氨酸残基评估了酶对各种底物的反应构象。 这些研究有助于更好地理解DNA修复途径，帮助细胞避免诱变并保持基因组完整性。 国家毒理学计划的一般毒理学和癌症小组一直在评估复杂混合物的毒理学特性，如膳食植物补充剂中发现的那些。 为此，NMR小组测量了来自不同制造商的银杏叶提取物中化合物的浓度，以评估相似性。 这些数据有助于比较各种技术方法，评估相似性，以获得最佳程序。 数据还表明，许多报告含有银杏的产品并不含有大量的银杏提取物。

Independent Project: Folate-dependent enzymes play a central role in single carbon metabolism and in nucleotide biosynthesis; its perturbation can be mutagenic and ultimately lethal. More generally, dysregulation of folate metabolism appears to be broadly linked to deficiencies in genome methylation, stability and repair. Alternatively, anti-folate drugs such as methotrexate are widely used in the treatment of neoplastic disease, but low doses have found increasing application in the treatment of rheumatoid diseases and other illnesses that are associated with chronic inflammation. Our previous studies of folate metabolism indicated that the NSAID fenbufen was able to interact with the enzyme dihydrofolate reductase (DHFR) by binding to the region of the folate binding site that interacts with the p-aminobenzoyl-L-glutamate (pABG). We are currently performing a more general assessment of the interaction of other NSAIDs with this subsite, their ability to bind to this site, and their inhibition of DHFR activity. These studies have quantified the DHFR inhibitory strength of various NSAIDs, and demonstrated that some possess even greater inhibitory potency than fenbufen. The potential anti-folate activity of NSAIDs suggests the possibility of additional applications in the treatment of chronic inflammatory diseases without some of the toxicity of more potent antifolate compounds such as methotrexate. NMR Core Activities: There are number of fluorinated anesthetics that are used in neurobiology, which can be difficult to solubilize. The NMR group utilized 19F NMR spectroscopy to assess the solution concentrations of several anesthetics for the Ion Channel Physiology Group in the Neurobiology Laboratory. The Stem Cell Biology Group in the Epigenetics and Stem Cell Biology Laboratory was working on new methods to create random mutagenesis by altering the chemistry of DNA bases. The NMR group utilized 1D NMR to assess the chemical structure and effectiveness of catalytic reactions to better optimize the procedures. The Macromolecular Structure Group in the Epigenetics and Stem Cell Biology Laboratory is looking at RNA structures to better understand how structural changes affect RNA regulatory pathways. The NMR group has been acquiring NMR data to determine the structure of the RNA targets. The Nuceolar Integrity Group in the Signal Transduction Laboratory is trying to understand the molecules involved in the pathway of ribosome biogenesis. The NMR group acquired data with the goal of providing information on how las1/grc3 interacts with RNA targets during biogenesis. The Pediatric Neuroendocrinology Group has been interested in how the protein SMCHD1, another chromatin remodeler, modulates ACE2 expression and to utilize our structural biology program to determine if SMCHD1 may be a druggable target. Downregulation of ACE2 expression is considered a potential treatment for SARS-CoV-2 infection. The NMR group has acquired data on conformations of SMCHD1 and are helping preparing samples for this project. The Matrix Biology group has been interested in the interactions of hyaluronic-acid (HA) with the RBD of the SARS-CoV-2 spike protein. The NMR group has run assays to assess the binding of different size HA with RBD and spike protein constructs. If HA blocks RBD binding of the spike protein of SARS-CoV-2 to ACE2, it may be a useful therapeutic. The Mutagenesis and DNA Repair Regulation Group is interested in how oxidative damage leads to mutagenesis. The group is studying now various yeast strains, and mutated yeast strain respond to oxidative chemical agents. In order to better understand the metabolic implications and how chemical pathways are affected by the oxidative chemical agents, the NMR group has been assessing the yeast metabolome from the strains and treatment groups. Metabolic pathways were assessed for similarities and differences for comparison with mutagenic profiles of the oxidative agents to better understand the chemical mechanisms of mutagenesis. The Mitochondrial DNA Replication Group tries to understand factors influence mutagenesis in the only organelle to maintain its own genome. One factor may be the formation of TT dimers due to UV irradiation near proposed G quadraplexes in the mitochondrial genome. The NMR group collected chemical data to assess the extent of TT dimer formation in different DNA samples based on sequence context and UV dose. The data was useful in optimizing further experiments to measure rates of mutagenesis. The Structural Cell Biology group is interested in how DNA damage is recognized and repaired. One important protein in sensing DNA damage is APTX. The NMR group has measured reaction rates for APTX under various conditions, and assessed the conformation of the enzyme in response to various substrates by examining the histidine residues. These studies contribute to a better understand of DNA repair pathways that help cells avoid mutagenesis and maintain genomic integrity. The General Toxicology and Cancer Group of the National Toxicology program has been assessing the toxicological properties of complex mixtures, like those found in dietary botanical supplements. Toward this end, the NMR group measured the concentrations of compounds in Ginkgo biloba extracts from various manufacturers in order to assess the similarity. The data was useful in comparing various technical methods, which evaluate the similarity, for the best procedure. The data also demonstrated that many products reported to contain Ginkgo biloba did not contain a significant amount of Ginkgo biloba extract.