Proton Loading Clusters and Complex Proton Pathways in Proton Pumping Proteins

生物体通过在线粒体或细菌细胞的膜的每一侧具有不同浓度的氢离子（H+）来储存代谢能量。在这个项目中要研究的蛋白质被嵌入这些膜中，从低浓度的一侧吸收H+，并将它们“泵”到高浓度的一侧。来自高浓度储存器的H+用于为基本的生物过程提供燃料，例如ATP的产生，ATP为许多细胞反应提供化学能。几种H+泵蛋白中原子的位置是已知的。该项目使用计算机模拟来研究：（1）蛋白质内部如何在对H+具有高或低亲和力之间切换，以便它可以首先结合，然后释放;（2）H+通过蛋白质内部的途径;（3）来自不同生物体的蛋白质如何保存它们的功能，即使蛋白质通过进化发生变化。该项目还将开发新的计算工具，这些工具，源代码，手册和视频说明将免费公开。研究项目将由本科生，硕士和博士生进行，为下一代科学家提供基础广泛的跨学科培训。这项研究的结果将被整合到一个基于项目的课程中，面向从工程到生物学的所有STEM领域的学生，向学生介绍蛋白质的结构如何决定其功能。这项研究将在纽约进行，该学院长期以来一直是不同背景学生通往中产阶级的途径。复合物I是第一种蛋白质，细胞色素c氧化酶（CCo）是最后一种，在有氧电子转移链中，利用释放能量的氧化还原反应将H+离子（质子）从较低（N-侧）驱动到较高（P-侧）。侧）膜的浓缩侧。这些蛋白质使用非常不同的结构来完成它们的任务，但都含有三个质子转移元件：（1）充满水的通道，由酸性、碱性和极性氨基酸侧链锚定，质子通过该通道移动;质子通过氢键连接从一个水或侧链传递到下一个;（2）质子装载位点（PLS）沿着路径，其是瞬时质子结合位点;这些位点在具有高质子亲和力以加载质子到低亲和力以释放质子之间变化;（3）当PLS加载时允许路径向N侧开放并且当其卸载时向P侧开放的门。水流出路径是关闭大门的一种方式。质子泵由它们的反应循环驱动以在质子转移状态之间移动，其中PLS加载或卸载并且门打开或关闭。本研究项目将调查这些构象变化。Gunner实验室开发的多构象连续静电（MCCE）方法将与分子动力学和网络分析相结合。MCCE在Monte Carlo计算中计算质子化微观态分布以及侧链和水氢键。质子化微观状态定义了每个残基的质子化状态。PLS被确定为改变质子化状态的残基，并且网络分析在混乱的氢键中发现质子转移路径。不同残基质子化和辅因子氧化还原状态的分子动力学模拟改变蛋白质构象。来自不同生物体的复合物I和细胞色素c氧化酶的序列和结构比较表明，组成PTS的残基及其位置可以改变。该项目将阐明多个CcO和复合物I中具有相同功能但低序列同一性的这些元件。更好地了解质子转移的范围和灵活性的要求，可以提供方法来控制许多生化反应，获得或失去质子在掩埋的活性位点。该研究由生物科学理事会分子和细胞生物科学部的分子生物物理学项目资助。该奖项反映了NSF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。

Living organisms store metabolic energy by having different concentrations of hydrogen ions (H+), on each side of the membranes of mitochondria or bacterial cells. The proteins to be studied in this project are embedded in these membranes, taking H+ from the side at lower concentration and ‘pumping’ them to the side at higher concentration. H+ from the high concentration reservoir is used to fuel essential biological processes such as the production of ATP, which supplies chemical energy for many cellular reactions. The positions of the atoms in several H+ pumping proteins are known. This project uses computer simulations to investigate: (1) How the protein interior can switch between having a high or low affinity for H+ so it can be first bound and then released; (2) The pathway the H+ take through the protein interior; and (3) How proteins from different organisms conserve their function, even as the proteins change through evolution. The project will also develop novel computational tools and these tools, source code, manuals and video instructions will be made freely, publicly available. The research projects will be carried out by undergraduates, masters and PhD students, leading to a broad-based, interdisciplinary training for the next generation of scientists. The findings of this research will be integrated into a project-based class for students majoring in all STEM fields, from engineering to biology, to introduce students on how the structure of a protein determines its function. The research will be carried out at the City College of New York, a college that has a long-standing, proven record as a pathway to the middle class for students from diverse backgrounds.Complex I is the first protein, and cytochrome c oxidase (CCo) the last, in the aerobic electron transfer chain that uses energy-releasing redox reactions to drive H+ ions (protons) from the lower (N-side) to the higher (P-side) concentration side of the membrane. These proteins use very different structures to accomplish their task but both contain three proton transfer elements: (1) Water filled channels, anchored by acidic, basic and polar amino acid side chains, through which protons move; protons pass via hydrogen bonded connections from one water or side chain to the next; (2) Proton Loading Sites (PLS) along the path, which are transient proton binding sites; These site change between having a high proton affinity to load protons to a low affinity one to release them; (3) Gates that allow the path to be open to the N-side when the PLS loads and are open to the P-side when it unloads. Water moving out of a path is one way to close a gate. Proton pumps are driven by their reaction cycle to move between proton transfer states with PLS loaded or unloaded and gates open or closed. This research project will investigate these conformational changes. The multi-conformation continuum electrostatics (MCCE) method, developed in the Gunner lab, will be integrated with molecular dynamics and network analysis. MCCE calculates the distribution of protonation microstates and the side chain and water hydrogen bonds in Monte Carlo calculations. A protonation microstate defines the protonation state of every residue. A PLS is identified as residues that change protonation state and network analysis finds proton transfer paths in the jumble of hydrogen bonds. Molecular dynamics simulations with different residue protonation and cofactor redox states change the protein conformations. Sequence and structure comparisons of Complex I and Cytochrome c oxidase from different organisms have shown that the residues that make up the PTS and their location can change. The project will elucidate these elements in multiple CcO and Complex I that have the same function but low sequence identity. Better understanding of the range and flexibility of requirements for proton transfer can provide methods to control the many biochemical reactions that gain or lose protons at buried active sites. This research is funded by the Molecular Biophysics program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences.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.