Probing the energy landscape of conformational changes in membrane proteins

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，所述动力学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，这些比率主要由某一事件发生的概率决定，而不是由运动本身决定。在运动过程中形成和破坏的相互作用的信息隐藏在集合测量中。 在这项研究中，我们的目标是解决这个“精细结构”的能量景观的结构重排，寡聚化和蛋白质折叠使用荧光光谱从平面脂质双层。由于事件之间缺乏同步，通常无法访问此信息。在刺激之后，每个事件都有一定的发生概率。因此，开始时间在几毫秒内呈指数分布。实际事件可能要快得多（1 ns-1us）。我们将通过研究单分子荧光或测量观察跃迁的参数来规避这个问题，而不是开始和最终状态。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的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）解决易位的速度并确定复杂膜蛋白的旋转运动。 通过X射线晶体学，我们已经有了蛋白质的原子尺度图像，包括膜蛋白。然而，为了能够安全地操纵它们的功能，用新药治疗遗传性疾病或细菌毒素，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。结果将允许直接分配两个氨基酸之间的单个相互作用的动态中的事件。

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.