Molecular Mechanisms of Active Transport Across Cellular Membranes

描述（由申请人提供）：膜转运蛋白是跨细胞膜的物质主动交换的主要参与者，这是活细胞中最基本和高度调节的过程之一。因此，研究其功能的分子基础在生物学和生物医学研究的各个学科中至关重要。这些复杂的蛋白质提供了高度复杂的、操作上微调的分子机器，以有效地将细胞中的各种能量源与各种分子种类逆着其化学梯度跨膜的矢量易位偶联。膜转运蛋白的机制的分子细节的调查构成了一个重大的挑战，主要是由于其结构的复杂性和高维度的能量依赖性的运输过程中提供的。底物结合和沿膜转运蛋白中渗透途径的沿着易位与由能量提供机制诱导和/或协调的各种量级的许多逐步蛋白质构象变化密切相关，质子和其它离子的结合和共转运，或ATP的结合和水解。因此，对膜转运蛋白中转运机制的详细描述依赖于能够在原子水平上描述这些过程的动力学的方法。分子动力学模拟仍然是一个高度相关的方法，具有足够的时间和空间分辨率来研究这些过程。膜转运蛋白的方法的应用是一个非常年轻的研究领域，因为足够的结构数据所需的这种模拟已经成为最近才可用。此外，为了描述涉及膜转运蛋白功能的生物学相关事件和步骤，需要对这些大生物分子进行至少0.1-1 μ s量级的原子模拟，这也是最近才得到满足的计算需求。虽然仍然极具挑战性，由于及时收敛的膜转运蛋白结构的发现和计算机硬件和软件的进步，我们现在在一个前所未有的位置，扩大范围的模拟研究到膜转运蛋白的领域，并调查其功能的分子基础。在这个应用中，我们提出了研究三种活性膜转运蛋白的项目：（1）麦芽糖转运蛋白，一种ABC转运蛋白，其中ATP结合和水解驱动底物转运过程;（2）谷氨酸转运蛋白（GluT），代表二级转运蛋白，其使用跨膜离子梯度作为其功能的能量来源;（3）线粒体ADP/ATP载体（AAC），它依赖膜电位在细胞质和线粒体之间进行核苷酸交换。公共卫生相关性：膜转运蛋白是介导多种物质选择性转运的蛋白质，例如，营养素、激素和神经递质，穿过细胞膜。它们对人体生理的几乎所有方面都是必不可少的，它们的功能障碍与大量的人类疾病有关。本申请中提出的研究将在分子水平上研究几种膜转运蛋白的功能机制。

DESCRIPTION (provided by applicant): Membrane transporters are principal players in active exchange of materials across the cellular membrane, one of the most fundamental and highly regulated processes in living cells. Investigating the molecular basis of their function, therefore, is of utmost importance in various disciplines of biological and biomedical research. These complex proteins provide highly sophisticated, operationally fine-tuned molecular machines to efficiently couple various sources of energy in the cell to vectorial translocation of various molecular species across the membrane against their chemical gradient. Investigation of the molecular details of the mechanism of membrane transporters poses a major challenge, primarily due to their structural complexity and the high dimensionality of the process of energy-dependent transport furnished by them. Substrate binding and translocation along the permeation pathway in membrane transporters is closely coupled to numerous stepwise protein conformational changes of various magnitudes that are induced and/or coordinated by the energy- providing mechanisms, e.g., binding and co-transport of protons and other ions, or binding and hydrolysis of ATP. A detailed description of the mechanism of transport in membrane transporters, therefore, relies on methods that can describe the dynamics of these processes at an atomic level. Molecular dynamics simulation remains a highly relevant approach with sufficient temporal and spatial resolutions to investigate such processes. Application of the method to membrane transporters is a very young area of research, since sufficient structural data required for such simulations has become available only recently. Furthermore, in order to describe biologically relevant events and steps involved in the function of membrane transporters, atomistic simulations of these large biomolecules on the orders of at least 0.1-1 <s are required, a computational demand which has also been met only recently. Though still extremely challenging, owing to the timely convergence of discoveries of membrane transporter structures and advances in computer hardware and software, we are now in an unprecedented position to expand the scope of simulation studies into the realm of membrane transporters and investigate the molecular basis of their function. In this application, we propose projects investigating three active membrane transporters: (1) Maltose transporter, an ABC transporter in which ATP binding and hydrolysis drive the process of substrate transport; (2) Glutamate transporter (GluT), representing secondary transporters that use the ionic gradient across the membrane as the source of energy for their function; and (3) the mitochondrial ADP/ATP carrier (AAC) which relies on the membrane potential for exchange of nucleotides betwee the cytoplasm and the mitochondria. PUBLIC HEALTH RELEVANCE: Membrane transporters are proteins that mediate selective transport of a wide range of materials, e.g., nutrients, hormones, and neurotransmitters, across the cellular membrane. They are essential to almost all aspects of human physiology, and their malfunction is associated with a large number of human diseases. The research proposed in this application will investigate the mechanism of function of several membrane transporters at a molecular level.