CAREER: Spectroscopic Studies of Functionally Significant Interactions in Iron-dependent Proteins and Enzymes

职业：铁依赖性蛋白质和酶中具有重要功能的相互作用的光谱研究

• 批准号: 2144239
• 负责人: Katlyn Meier
• 金额: $ 67万
• 依托单位: University of Miami
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2144239&HistoricalAwards=false
• 关键词: CAREER; Spectroscopic; Studies; Functionally; Significant

With this CAREER award, the Chemistry of Life Processes Program in the Chemistry Division is funding Katlyn Meier from the University of Miami to investigate protein-protein interactions in iron-containing metalloproteins and metalloenzymes using a metal-centric spectroscopy approach. Protein-protein interactions define how proteins bind to and interact with each other to affect their structures and how they function. Dr. Meier’s research will study the interactions involving two different proteins that contain iron by using methods that follow specifically the chemical properties of this metal at the protein-protein interaction surface. These spectroscopic methods, when coupled with computation, will provide information on interaction interfaces among proteins under conditions that are less artificial and more similar to those found in cells. One of these iron containing proteins interacts with other similar proteins to control the synthesis of important molecules, including vitamins and hormones, or inhibit metabolism of drugs. The second iron protein being studied assembles into a complex composed of four identical copies of itself. This tetramer protein is a catalyst that helps produce compounds that reduce effects of oxidants in cells. The project will provide training to graduate and undergraduate students in advanced biochemical, biophysical, and computational methods. Finally, the project includes an integrated outreach program aimed at introducing middle and high school students to the basics of spectroscopy that includes a mentorship program aimed at increasing retention of future scientists from underserved populations.Characterization of the protein-protein interaction (PPI) interface and the role of the metals requires an interdisciplinary approach that employs several spectroscopic techniques. Low temperature electron paramagnetic resonance (EPR) spectroscopy will be used to selectively interrogate the structural and electronic properties of the iron sites and to provide feedback on the paramagnetic species in each sample. Variable temperature, variable field Mössbauer spectroscopy will be used to conclusively determine iron or heme oxidation state and to measure coupling between iron centers. Changes in secondary and quaternary structures that occur as the result of protein conformational changes induced by the PPI will be monitored by far-UV circular dichroism (CD), while CD in the visible and near-infrared regions will report on changes in charge transfer bands and d-d centered transitions relevant to perturbations in the metal-protein complex. Finally, fluorescence measurements will provide additional feedback about the interaction surface via intrinsic tryptophan fluorescence quenching, among other methods. The combination of these techniques is expected to provide for greater understanding of global and local electronic and structural changes associated with PPIs by spanning a wide range of energy scales that, in turn, probe conformational changes at various distance scales. Insight gained from this work will be used to calibrate computational models of PPIs. It is anticipated that the proposed studies will provide spectroscopically informed insight that is currently lacking in the field, but yet is required for “bottom-up” design of peptidomimetics to modulate and report on PPIs.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.

凭借这一职业奖，化学部门的生命过程化学项目资助了迈阿密大学的Katlyn Meier，利用金属中心光谱方法研究含铁金属蛋白和金属酶中的蛋白质-蛋白质相互作用。蛋白质之间的相互作用定义了蛋白质如何结合并相互作用，从而影响它们的结构和功能。Meier博士的研究将研究两种含有铁的不同蛋白质之间的相互作用，具体方法是根据蛋白质-蛋白质相互作用表面金属的化学性质。这些光谱方法，当与计算相结合时，将提供蛋白质之间相互作用界面的信息，在更少人为和更类似于细胞中发现的条件下。其中一种含铁蛋白质与其他类似蛋白质相互作用，控制重要分子的合成，包括维生素和激素，或抑制药物的代谢。第二个被研究的铁蛋白组装成一个复合体，由四个相同的自身副本组成。这种四聚体蛋白是一种催化剂，有助于产生减少细胞中氧化剂影响的化合物。该项目将为研究生和本科生提供高级生化、生物物理和计算方法方面的培训。最后，该项目还包括一个综合推广计划，旨在向初高中学生介绍光谱学的基础知识，其中包括一个指导计划，旨在从服务不足的人群中增加未来科学家的留用。表征蛋白质-蛋白质相互作用（PPI）界面和金属的作用需要采用多种光谱技术的跨学科方法。低温电子顺磁共振（EPR）光谱将用于选择性地询问铁位点的结构和电子性质，并提供每个样品中顺磁物质的反馈。变温度，变场Mössbauer光谱将用于确定铁或血红素氧化状态，并测量铁中心之间的耦合。由PPI引起的蛋白质构象变化导致的二级和四级结构的变化将通过远紫外圆二色性（CD）进行监测，而可见光和近红外区的CD将报告与金属-蛋白质复合物中扰动相关的电荷转移带和d-d中心跃迁的变化。最后，荧光测量将通过色氨酸荧光猝灭等方法提供关于相互作用表面的额外反馈。这些技术的结合有望通过跨越大范围的能量尺度，从而在不同距离尺度上探测构象变化，从而更好地了解与ppi相关的全局和局部电子和结构变化。从这项工作中获得的见解将用于校准ppi的计算模型。预计拟议的研究将提供目前在该领域缺乏的光谱信息，但仍需要“自下而上”的肽模拟物设计来调节和报告ppi。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。