Time-resolved conformational changes of proteins by very high frequency Gd3+ EPR

这个项目旨在开发一种方法来“拍摄”蛋白质，这种微小的机器使我们所知的所有生命在履行其关键的生物功能时得以实现。了解蛋白质的功能是最令人兴奋的科学前沿之一，为了满足可再生能源和农业等各种领域的社会需求，了解蛋白质的功能是必要的。由于X射线光谱、磁共振和其他方法的革命性进步，超过10万种不同类型的蛋白质的丰富和复杂的三维形状现在被记录在案。然而，尽管知道机器的形状，例如，看到缝纫机的照片，可能会给人们提供一个关于机器如何工作的线索，但一部机器运行的电影要揭示得多。这个项目的目标是开发新的方法来制作这样的蛋白质电影，或者更准确地说，开发方法来测量蛋白质结构(或结构动力学)中的时间分辨构象变化。一个由研究生和本科生组成的团队，致力于物理、化学和生物之间的相互作用，并与国际合作者密切合作，将解决这一令人兴奋的问题，并将成为美国科学、技术、工程和数学(STEM)劳动力的领导者。要想拍摄起作用的蛋白质，需要一种校准的工具，以高通量测量几纳米的距离，理想情况下，时间分辨率为亚毫秒级。该工具必须与蛋白质执行其功能的复杂局部环境兼容；理想情况下，在水溶液中，尽管许多蛋白质只要在约215K以上就继续发挥作用。这项研究的主要目标是使用高频(200 GHz)电子顺磁共振(EPR)结合具有自旋-7/2 Gd3+部分的定点自旋标记来测量蛋白质的时间分辨构象变化。在之前的NSF支持下，PI和共同PI的合作已经证明，最简单的240 GHz EPR测量；包含一对Gd3+自旋标签的分子线形的测量；即使在室温下也能够分辨大于3 nm的距离。拟议的研究将以这些关键发展为基础，主要侧重于蛋白质蛋白紫质模型的时间分辨距离测量，这是一种光合作用跨膜质子泵，可以在不到1微秒到1秒以上的时间尺度上触发光合作用跨膜质子泵，开始同步构象变化的级联。主要工作是：(1)发展一种基于测量Gd3+EPR谱线线形的简单方法，从Gd3+双标记的PR中提取1.5-4 nm范围内的距离和距离分布。(2)以亚毫秒级的时间分辨率测量PR的构象变化，温度范围从大约215K(PR处于冻结状态，但处于活动状态)到室温。(3)Gd3+的脉冲EPR研究，以了解Gd3+在很高频率下的自旋物理。

This project aims to develop a method to "film" proteins, the tiny machines that enable all of life as we know it, as they perform their critical biological functions. Understanding how proteins function is one of the most exciting frontiers of science, and is necessary in order to address societal needs in fields as diverse as renewable energy and agriculture. The rich and intricate 3-dimensional shapes of more than 100,000 different kinds of proteins are now documented, thanks to revolutionary advances in X-ray spectroscopy, magnetic resonance, and other methods. However, while knowing the shape of a machine, for example, seeing a photograph of a sewing machine, might give one a clue as to how it works, a movie of a machine in action is far more revealing. The goal of this project is to develop new methods to make such "movies" of proteins, or stated more precisely, to develop methods to measure the time resolved conformational changes in protein structure (or structural dynamics). A team of graduate students and undergraduates, working at the interface between physics, chemistry, and biology, and working closely with international collaborators, will tackle this exciting problem, and will emerge well-positioned to become leaders in the nation's science, technology, engineering and mathematics (STEM) workforce."Filming" proteins in action requires a calibrated tool to measure distances of several nanometers with high throughput and, ideally, sub-millisecond time resolution. This tool must be compatible with the complex local environments in which proteins perform their functions; ideally, in aqueous solutions although many proteins continue to function as long as they are above about 215K. The primary goal of the proposed research is to use high-frequency (200 GHz) electron paramagnetic resonance (EPR) combined with site-directed spin labeling with spin-7/2 Gd3+ moieties to measure time-resolved conformational changes of proteins. With prior NSF support, the PI and co-PI's collaboration has demonstrated that the simplest possible 240 GHz EPR measurements; measurements of the lineshapes of molecules containing a pair of Gd3+ spin labels; is capable of resolving distances greater than 3 nm even at room temperature. The proposed research will build on these key developments, focusing primarily on time-resolved distance measurements in the model protein Proteorhodopsin (PR), a photosynthetic trans-membrane proton pump that can be triggered with flashes of light to begin synchronized cascades of conformational changes on time scales ranging from less than 1 microsecond to more than 1 second. The main activities undertaken will be: (1) Developing a simple method based on measuring the lineshape of the Gd3+ EPR line to extract distances and distance distributions in the 1.5-4 nm range from PR doubly labeled with Gd3+. (2) Measuring conformational changes of PR at temperatures ranging from about 215K (where PR is frozen but active) to room temperature with sub-millisecond time resolution. (3) Pulsed EPR studies of Gd3+ to understand the spin physics of Gd3+ at very high frequencies.