CAREER: Research and education on protein folding as an energy source at the bacterial outer membrane

所有的革兰氏阴性细菌都有两层不同的膜围绕着它们。这些膜含有大量的膜蛋白。在内膜中，大多数蛋白质采用被称为α-螺旋折叠的螺旋形式，而在外膜中，它们几乎都是通过蛋白质链的并排排列形成片状结构，这被称为β-折叠。外膜含有具有大而开放的孔的蛋白质，使其无法包含储存在小分子中的化学能量，如ATP或以离子梯度的形式储存。因此，复杂的过程需要一种替代能源，如将蛋白质插入膜或通过膜分泌蛋白质。这个项目的目标是确定蛋白质折叠本身作为能量来源的可行性。这个项目试图改变高中生物教学中的一种常见范式，在这种范式中，细胞和包含在其中的系统通常被视为静态和高度定向的。通过模拟数据的分子可视化，蛋白质的动态性质变得显而易见。将在北乔治亚州地区的高中教室中开发和实施关于特定主题的多个单元，例如膜的组装和通过膜结合通道的扩散。这些模块强调为观察到的行为构建解释性模型，这是未来科学标准的关键要素。它们的实施将得到可视化程序VMD的简化界面开发的帮助。这些单元的影响评估将首先在三个教室进行，然后通过网络分发扩大到更广泛的受众。研究生和本科生将高度参与整个过程，为他们提供不仅仅是研究的培训。已经提出了多种理论模型来解释蛋白质折叠形成β-折叠是如何提供能量的。在这项建议中，将进行计算研究，以确定通过蛋白质折叠形成这些结构的能量方面(1)分离，(2)插入外膜结合的蛋白质和(3)跨膜的自动转运体结构域的分泌。对这些过程的能量方面的定量测定将用于评估和改进现有的模型。对两个特定系统的研究将用于阐明BAMA是如何插入外膜蛋白的，以及自体转运蛋白是如何分泌毒力蛋白结构域的，两者都是通过自己的折叠来提供能量的。具体目标是(1)表征自由能景观和β-折叠结构的稳定性；(2)确定BAMA如何催化外膜蛋白质的插入和折叠；(3)解决自动转运体折叠中发生的分子序列；以及(4)将生物系统的动力学整合到高中教学中。采用的主要方法是分子动力学(MD)模拟。所有的模拟结果都将与实验紧密结合，既有回溯性的，也有前瞻性的。

All Gram-negative bacteria possess two distinct membranes surrounding them. These membranes contain numerous membrane proteins. In the inner membrane, most proteins adopt a helical form termed alpha-helical fold, whereas in the outer membrane they almost all form sheet-like structures by side-by-side alignment of protein strands, which is termed beta-sheet fold. The outer membrane contains proteins with large, open pores, rendering it unable to contain chemical energy stored in small molecules such as ATP or in the form of an ion gradient. Therefore, an alternative source of energy is required for complex processes such as insertion of proteins to membrane or secretion of proteins through the membrane. It is the goal of this project to determine the feasibility of protein folding itself as the source of energy. This project seeks to change a common paradigm in high school biology instruction, in which cells and the systems contained within are often seen as static and highly directed. Through molecular visualization of simulation data, the dynamic nature of proteins becomes readily apparent. Multiple modules on specific topics, such as the assembly of membranes and diffusion through membrane-bound channels, will be developed and implemented in high school classrooms in the North Georgia area. These modules emphasize the construction of explanatory models for the observed behavior, a key element of future science standards. Their implementation will be aided by the development of a simplified interface for the visualization program VMD. Assessment of the modules' impact will first be conducted in three classrooms before expanding to a wider audience through web distribution. Graduate and undergraduate students will be highly involved in the entire process, giving them training beyond just research. Multiple theoretical models have been proposed to explain how the folding of proteins to form beta-sheet can provide energy. In this proposal, computational investigations will be performed to determine, the energetic aspects of the formation of these structures through protein folding (1) in isolation, (2) in the insertion of outer-membrane-bound proteins and (3) in the secretion of auto transporter domains across the membrane. Quantitative determination of the energetic aspects of these processes will be used to evaluate and refine existing models. Investigations into two specific systems will be used to elucidate how outer-membrane proteins are inserted by BamA and how virulence protein domains are secreted by autotransporters, both being energized by their own folding. The specific objectives are to (1) characterize the free-energy landscape and stability of beta-sheet structures; (2) determine how BamA catalyzes the insertion and folding of outer-membrane proteins; (3) resolve the molecular sequence of events in autotransporter folding; and (4) integrate dynamics of biological systems into high school instruction. The primary method to be used is molecular dynamics (MD) simulation. All simulation results will be closely coupled to experiments, both retrospectively and prospectively.