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

通过这项职业奖，化学部生命过程化学项目资助迈阿密大学的Katlyn Meier使用以金属为中心的光谱学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-蛋白质相互作用定义了蛋白质如何相互结合和相互作用，从而影响它们的结构和功能。梅尔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，方法是特别遵循这种金属在蛋白质-蛋白质相互作用表面的化学性质。这些光谱方法，当与计算相结合时，将提供关于蛋白质之间相互作用界面的信息，这些条件不那么人为，更类似于细胞中的情况。这些含铁蛋白质中的一种与其他类似的蛋白质相互作用，以控制包括维生素和激素在内的重要分子的合成，或抑制药物的新陈代谢。正在研究的第二种铁蛋白组装成一个由四个完全相同的自身副本组成的复合体。这种四聚体蛋白质是一种催化剂，有助于产生减少细胞内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，该项目包括一个旨在向初中生介绍光谱学基础知识的综合推广计划，其中包括一个旨在增加未来科学家从服务不足的人群中留住的导师计划。蛋白质-蛋白质相互作用(PPI)界面的特征和金属的作用需要使用多种光谱技术的跨学科方法。低温电子顺磁共振(EPR)谱将被用来选择性地询问铁位置的结构和电子性质，并提供关于每个样品中顺磁物种的反馈。变温、变场穆斯堡尔光谱分析将用于最终确定铁或血红素的氧化状态，并测量铁中心之间的耦合。由于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谱(CD)来监测，而在可见光和近红外区域的CD将报告与金属-蛋白质络合物中的扰动相关的电荷转移带和d-d中心跃迁的变化。最后，荧光测量将通过固有的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。这些技术的结合预计将通过跨越广泛的能量尺度，进而探测不同距离尺度的构象变化，从而更好地了解与PPI相关的全球和局部电子和结构变化。从这项工作中获得的洞察力将用于校准PPI的计算模型。预计拟议的研究将提供该领域目前缺乏的光谱信息洞察力，但却是模拟多肽的“自下而上”设计所必需的，以调节和报告PPIs。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.