Protein Interactions in the Utilization of Iron by Bacteria

智力优势：铁是一种必需的营养素，参与基本的细胞过程，从氧气储存和运输到呼吸和DNA修复。病原菌具有铁吸收机制，使它们能够利用由乳铁蛋白、转铁蛋白或结合血红素螯合的宿主铁，从而克服其宿主生物体中遇到的低铁浓度。当铁到达细菌胞质溶胶时，它的命运知之甚少。铁蛋白分子作为细胞溶质铁浓度的动态调节剂发挥作用，但尽管它们在铁稳态中的重要性，但对提供Fe 2+用于储存的过程或促使其释放以安全整合在代谢中的信号知之甚少。铜绿假单胞菌中有两种铁蛋白样分子：细菌铁蛋白（BfrB），由24个相同的亚基组装而成，并结合12个血红素分子;细菌铁蛋白（FtnA），也由24个相同的亚基组装而成，但不结合血红素。最近已经发现，铁从BfrB的释放需要电子由铁氧还蛋白（Bfd）和铁氧还蛋白还原酶（FPR）介导，而铁从FtnA的释放需要电子仅由FPR介导。这些发现提供了一个独特的平台，研究蛋白质结构和蛋白质-蛋白质相互作用如何有助于致病菌胞质铁的调节。为了研究这些蛋白质-蛋白质相互作用，将采用多学科方法，包括生物化学，NMR光谱学，X射线晶体学和计算生物学。具体目标是：1）研究并从结构上确定调节铁从BfrB和FtnA内腔流出的蛋白质-蛋白质相互作用。(2)挑战一个假设，即Fe 2+退出BfrB和FtnA内腔通过四倍孔形成在其结构中的交叉点4-subunit.Broader影响：该项目的多学科性质将提供许多机会，为学生在所有学术水平。通过每月的联合小组会议，在里维拉，Vakser和Lovell实验室（均位于堪萨斯大学）的努力的整合将使学生接触到智力多样的氛围，这是培养多学科研究所必需的。这种经验，再加上三个实验室中丰富的种族环境（亚洲人，高加索人，西班牙裔;约40/60女性/男性）将为研究生毕业后可能遇到的多方面工作环境做好准备。同样重要的是，这个项目的合作精神将向学生说明一个基础广泛的方法的好处：（1）解决复杂的问题;（2）更有效地把在一个实验室获得的结果放在适当的背景下;从而（3）改善研究将影响更大的科学界的前景，该项目由分子和细胞生物科学部的生物分子动力学、结构和功能群以及化学部的生命过程化学项目共同支持。

Intellectual Merit: Iron is a required nutrient involved in fundamental cellular processes, from oxygen storage and transport to respiration and DNA repair. Pathogenic bacteria are equipped with iron uptake mechanisms that enable them to utilize host-iron sequestered by lactoferrin, transferrin or bound to heme, and therefore overcome the low-iron concentrations encountered in their host organisms. Much less is known about the fate of iron once it reaches the bacterial cytosol. Ferritin molecules function as dynamic regulators of cytosolic iron concentrations, but despite their importance in iron homeostasis, little is known about the processes that deliver Fe2+ for storage or the signals that prompt its release for safe integration in metabolism. There are two ferritin-like molecules in P. aeruginosa: a bacterioferritin (BfrB), which assembles from 24 identical subunits and binds 12 heme molecules, and a bacterial ferritin (FtnA), which is also assembled from 24 identical subunits but does not bind heme. It has recently been discovered that release of iron from BfrB requires electrons to be mediated by a ferredoxin (Bfd) and a ferredoxin reductase (FPR), whereas release of iron from FtnA requires that electrons be mediated only by FPR. These findings provide a unique platform to study how protein structure and protein-protein interactions contribute to the regulation of cytosolic iron in pathogenic bacteria. In order to investigate these protein-protein interactions a multidisciplinary approach will be undertaken which includes biochemistry, NMR spectroscopy, X-ray crystallography and computational biology. The specific goals are: 1) Investigate and structurally determine the protein-protein interactions that regulate the outflow of iron from the interior cavities of BfrB and FtnA. (2) Challenge a hypothesis that Fe2+ exits the BfrB and FtnA interior cavities via the four-fold pores formed in their structures at the intersection of 4-subunits.Broader Impacts: The multidisciplinary nature of the project will provide many opportunities for students at all academic levels. The integration of efforts in the Rivera, Vakser and Lovell laboratories (all located at the University of Kansas) via monthly joint group meetings will expose the students to the intellectually diverse atmosphere that is necessary to nurture multidisciplinary research. This experience, coupled to the ethnically rich environment present in the three labs (Asian, Caucasian, Hispanic; ~40/60 female/male) will prepare the graduate students well for the multifaceted work environment they are likely to encounter after graduation. Just as important, the collaborative spirit of this project will illustrate to students the benefits of a broad-based approach for (1) solving complex problems; (2) more effectively putting the results obtained in one laboratory into its proper context; and thus (3) improving the prospect that the research will impact the greater scientific community, and ultimately benefit the general public.This project is jointly supported by the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.