Probing the energy landscape of conformational changes in membrane proteins

探索膜蛋白构象变化的能量景观

• 批准号: 327201-2012
• 负责人: Blunck, Rikard
• 金额: $ 3.28万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=571949
• 关键词: Probing; energy; landscape; conformational; changes

The function of membrane proteins is often related to a structural rearrangement. These may be described by kinetic models that provide macroscopic transition rates for the transition from one conformation to another. However, these rates are mainly determined by the probability that a certain event occurs, not by the movement itself. The information about interactions that form and break during the movement is hidden in ensemble measurements. In this research proposal, we aim to resolve this "fine structure" of the energy landscape for structural rearrangements, oligomerization and protein folding using fluorescence spectroscopy from planar lipid bilayers. This information is in general not accessible due to the lack of synchronization between events. Following a stimulus, each event has a certain probability to occur. The start times are thus exponentially distributed over several milliseconds. The actual events are likely much faster (1ns-1us). We will circumvent this problem by studying single molecule fluorescence or measuring a parameter that observes the transition, not the start and final state. We will thus investigate (1) the protein translocation through an anthrax pore, giving insight into single amino acid interactions and protein folding; (2) protein oligomerization in planar lipid bilayer; and (3) resolve the speed of translocations and determine rotational movements in complex membrane proteins. With x-ray crystallography, we already have atomic scale images of proteins including membrane proteins. Nevertheless, in order to be able to securely manipulate their function with new drugs for inherited diseases or bacterial toxins with less side effects, we need to understand the movement of the proteins and their relation to the atomic scale models. The results will allow to assign directly single interactions between two amino acids to an event in the dynamics.

膜蛋白的功能通常与结构重排有关。这些可以通过动力学模型来描述，该模型提供从一种构象到另一种构象的转变的宏观转变速率。然而，这些比率主要由特定事件发生的概率决定，而不是由运动本身决定。有关运动过程中形成和破坏的相互作用的信息隐藏在整体测量中。 在这项研究计划中，我们的目标是利用平面脂质双层的荧光光谱来解决结构重排、寡聚和蛋白质折叠的能量景观的“精细结构”。由于事件之间缺乏同步，该信息通常无法访问。在刺激之后，每个事件都有一定的发生概率。因此，开始时间呈指数分布在几毫秒内。实际事件可能要快得多（1ns-1us）。我们将通过研究单分子荧光或测量观察转变而不是起始状态和最终状态的参数来规避这个问题。因此，我们将研究（1）通过炭疽孔的蛋白质易位，深入了解单个氨基酸相互作用和蛋白质折叠； (2)平面脂质双层中的蛋白质寡聚化； (3) 解析易位速度并确定复杂膜蛋白的旋转运动。 通过 X 射线晶体学，我们已经获得了包括膜蛋白在内的蛋白质的原子尺度图像。然而，为了能够用治疗遗传性疾病或细菌毒素的新药安全地操纵它们的功能，同时减少副作用，我们需要了解蛋白质的运动及其与原子尺度模型的关系。结果将允许将两个氨基酸之间的单一相互作用直接分配给动力学中的事件。