Proton Loading Clusters and Complex Proton Pathways in Proton Pumping Proteins

生物体通过线粒体或细菌细胞膜两侧不同浓度的氢离子（H+）来储存代谢能量。在这个项目中要研究的蛋白质嵌入在这些膜中，从低浓度的一侧吸收H+，并将它们“泵”到高浓度的一侧。来自高浓度储存库的H+用于为必要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。几个氢离子泵送蛋白的原子位置是已知的。本项目利用计算机模拟研究：(1)蛋白质内部如何在对H+具有高亲和力或低亲和力之间切换，从而使其首先结合然后释放；(2) H+通过蛋白质内部的途径；(3)即使蛋白质在进化过程中发生了变化，来自不同生物体的蛋白质如何保持它们的功能。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提供。这些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、跨学科的培训。这项研究的结果将被整合到从工程到生物学的所有STEM领域的学生的项目基础课程中，向学生介绍蛋白质的结构如何决定其功能。这项研究将在纽约城市学院（City College of New York）进行，这所学院长期以来一直被证明是来自不同背景的学生进入中产阶级的途径。在有氧电子传递链中，复合物I是第一个蛋白质，细胞色素c氧化酶（CCo）是最后一个蛋白质，它利用释放能量的氧化还原反应将H+离子（质子）从膜的低浓度（n侧）驱动到高浓度（p侧）。这些蛋白质使用非常不同的结构来完成它们的任务，但都包含三个质子转移元素：(1)由酸性、碱性和极性氨基酸侧链固定的充满水的通道，质子通过这些通道移动；质子通过氢键连接从一个水链或侧链传递到下一个；(2)路径上的质子加载位点（Proton Loading Sites， PLS），是瞬时质子结合位点；这些位点在具有高亲和力来装载质子和具有低亲和力来释放质子之间变化；(3)在PLS负载时允许通路向n侧打开，在PLS卸载时允许通路向p侧打开的门。水流出通道是关闭大门的一种方式。质子泵由其反应周期驱动，在质子转移状态之间移动，PLS加载或卸载，闸门打开或关闭。本研究项目将调查这些构象变化。由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在蒙特卡罗计算中计算质子化微态的分布以及侧链和水氢键。质子化微态决定了每个残基的质子化态。一个PLS被确定为改变质子化状态的残基，网络分析发现质子转移路径在混乱的氢键中。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了蛋白质的构象。不同生物的复合物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。该项目将阐明这些元素在多个CcO和复合体I具有相同的功能，但低序列同一性。更好地理解质子转移需求的范围和灵活性，可以提供控制许多生物化学反应的方法，这些反应会在埋藏的活性位点获得或失去质子。本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。

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.