CAREER: Secondary Active Membrane Transporters: Determining Protein Structure and Transport Mechanisms with a New Hybrid Simulation

智力优势细胞膜是所有生物有机体的重要组成部分。它们可以保护细胞或细胞内有自己的膜的隔间免受有害化合物的伤害。跨越细胞膜的膜运输蛋白可以充当把关人，控制有益分子的流入，并使有害分子能够外流。膜转运蛋白根据它们的功能被分成几类；其中一类，次级活性转运蛋白(SAT)，是这个项目特别感兴趣的。饱和脂肪酸将较小的初级底物(质子或离子)的运动与较大底物(糖、氨基酸、多肽)的运动相结合。这些蛋白质利用初级底物下坡运动(从高到低浓度)获得的能量，促进较大底物的上坡运动(从低浓度到高浓度)。虽然已经确定了几个SAT的原子级结构，但对于给定的蛋白质，通常只知道运输周期中的单一构象。然而，底物的运输涉及重大的蛋白质结构变化，这些变化不是单一构象所能捕捉到的。本研究的主要目的是用原子水平模拟来研究几种SAT的转运机制和多种蛋白质构象的变化。一种名为“隐式-显式膜模拟”的新的原子分辨率模拟方法将被用来加强构象采样，同时保持构象状态之间的自然跃迁。首先，这种新方法将在海因钠转运体(Mhp1)上进行测试，因为它的运输周期中的几个结构已经从X射线衍射中确定。在与实验者的合作中，将研究面向细胞内外的Mhp1构象之间的转变，以及稳定新结构的可能突变，例如面向内的闭塞结构。对Mhp1的研究将为将这种新方法用于其他SAT奠定基础，特别是那些只有一种已知蛋白质构象的SAT，如乳糖渗透酶。这一新的模拟工具最终将允许研究SAT中以前未知的运输周期状态，并使实验结构生物学家深入了解如何稳定这些状态。此外，对这些SAT的研究可能有助于更好地理解哺乳动物、植物和单细胞生物体中类似的蛋白质如何运输底物和改变构象。BROADER影响将开发高中教学辅助工具，以促进学生对分子生物学的积极参与和兴趣。具体地说，将开发一个教育网站，重点关注三个一般主题，即蛋白质、细胞膜和膜蛋白的功能。该网站将包括描述每个主题的文本、图形、电影和交互式小应用程序。此外，该项目将继续开发本科和研究生联合课程--分子建模方法，旨在向学生介绍分子模拟方法的化学和生物分子方面。此外，本科生和研究生，特别是那些在研究中代表性较低的背景的学生，将被招募参加计算生物学技术培训。实现这些教育目标的关键是正确评估教育网站、教学和辅导的影响。与一位在开发教育工具、评估和问卷方面经验丰富的教育研究人员合作，将有助于实现这些教育目标。总体而言，实施这一教育计划将提高高中生对STEM职业的兴趣，促进对计算生物学的兴趣，并培养下一代科学和工程研究人员。该项目得到了分子和细胞生物科学部细胞过程集群和化学系化学理论、模型和计算方法项目的联合支持。

INTELLECTUAL MERIT Cell membranes are a crucial component of all biological organisms. They can protect the cell or compartments within a cell that have their own membranes from harmful compounds. Membrane transport proteins that span cell membranes can act as gatekeepers that control the influx of helpful molecules and enable the efflux of harmful molecules. Membrane transport proteins are grouped into classes based on their function; one such class, secondary active transporters (SATs), is of specific interest to this project. SATs couple the movement of a small primary substrate (protons or ions) to that of a larger substrate (sugars, amino acids, peptides). These proteins use the energy gain from the downhill movement (high to low concentration) of the primary substrate to facilitate the uphill movement (low to high concentration) of a larger substrate. Although atomic-level structures have been determined for several SATs, for a given protein typically only a single conformation in the transport cycle is known. However, transport of substrates involves significant protein structural changes that are not captured by a single conformation. The main objective of this research is to investigate the transport mechanisms and multiple protein conformational changes of several SATs with atomic-level simulations. A new atomic resolution simulation approach known as "implicit-explicit membrane simulation" will be used to enhance conformational sampling while preserving the natural transition between conformational states in SATs. Initially, this new method will be tested on the sodium-hydantoin transporter (Mhp1) because several structures in its transport cycle have been determined from x-ray diffraction. In collaboration with experimentalists, transitions between Mhp1 conformations facing inside and outside the cell will be researched, as will possible mutations that stabilize new structures, such as the inward-facing occluded structure. Studies on Mhp1 will lay the groundwork for using this new method on other SATs, especially those with only a single known protein conformation, such as lactose permease. This new simulation tool ultimately will allow studies of previously unknown transport cycle states in SATs and give experimental structural biologists insight into how these states may be stabilized. Moreover, studies of these SATs may lead to better understanding of how similar proteins in mammals, plants, and single-celled organisms transport substrates and change conformations.BROADER IMPACTS High school instructional aids will be developed to promote active student engagement and interest in molecular biology. Specifically, an educational website will be developed that focuses on three general topics, namely, proteins, cell membranes, and function of membrane proteins. This website will include text, figures, movies, and interactive applets that describe each topic. In addition, this project will continue the development of a joint undergraduate and graduate course, Molecular Modeling Methods, intended to introduce students to chemical and biomolecular aspects of molecular simulation methods. Additionally, undergraduate and graduate students, particularly those from backgrounds underrepresented in research, will be recruited for training in computational biology techniques. Key to these educational goals is proper assessment of the impact of the educational website, teaching and mentoring. Collaboration with an educational researcher experienced in developing educational tools, assessments, and questionnaires will aid in fulfilling these educational goals. Overall, implementing this educational plan will increase high school students interest in STEM careers, promote interest in computational biology, and train the next generation of science and engineering researchers.This project is jointly supported by the Cellular Processes Cluster in the Division of Molecular and Cellular Biosciences and the Chemical Theory, Models and Computational Methods program in the Chemistry Division.