Robust Gd3+ -based spin labels for structural studies of membrane proteins

智力优势：活细胞内部和外部之间的边界是一层只有几纳米厚的膜。嵌入细胞膜的蛋白质是控制物质、能量和信息在细胞边界上流动的分子机器，了解它们的结构有助于了解它们的功能。不幸的是，像晶体学和核磁共振这样的结构技术并不适用于膜蛋白，所以需要新的方法。该研究项目将开发新的分子探针和实验方法，以提供有关蛋白质结构的信息。新的探针利用了元素钆（Gd3+）离子的显著磁性，钆离子已被用于增强磁共振成像（MRI）的对比度。当在非常高的磁场中被激光激发时，钆离子以每秒2400亿周期（240千兆赫）的频率像顶部一样进动（摆动）；这个过程大约会发生20000次，然后大部分时间会放松到热平衡。当一个Gd3+离子靠近另一个Gd3+离子时，它弛豫得更快。此外，当两个离子之间的距离小于10纳米时，进动振幅在两个离子之间以“节拍频率”振荡，该频率随离子之间的距离而敏感地变化。弛豫时间、岁差频率和节拍频率一起给出了距离（最终是结构）信息。这些参数将使用加州大学圣巴巴拉分校（240千兆赫兹）和以色列魏茨曼科学研究所（95千兆赫兹）新开发的电子顺磁共振（EPR）光谱仪进行测量。为了校准新方法，钆离子之间的距离测量将在“标尺”化合物上进行，这些化合物由Gd3+离子组成，由坚硬的线性聚合物隔开，已知距离在2到12纳米之间。然后在蛋白紫质上精心选择的位点之间进行距离测量。变形紫红质是一种从海洋细菌中分离出来的光激活质子泵，其结构与所有其他生物中的膜蛋白相似。更广泛的影响：该项目是美国（Song-I Han和Mark Sherwin，加州大学圣巴巴拉分校）、以色列（Daniella Goldfarb，魏茨曼科学研究所）和德国（Adelheidt Godt，比勒菲尔德大学，探针合成）之间的一个新的跨学科国际合作项目。合作者和共同负责人都是在开发新技术方面具有相当专业知识的女性物理学家。该提案将支持两名研究生和一名本科生。这些学生在汉实验室（物理化学和生物化学）和舍温实验室（实验物理）之间穿梭，接受交叉训练，因此沉浸在“软物质”、磁共振、蛋白质、微波电子学、太赫兹科学与技术和可见光学等科学领域。学生还将在以色列魏茨曼科学研究所著名的磁共振社区进行实验，通过国际科学经验极大地丰富他们的训练。学生们开发的Gd3+自旋标签和高场电子顺磁共振技术将使许多其他科学家能够在类生命环境中对膜蛋白进行新的研究。这些标签足够稳定，可以在复杂的活细胞环境中使用，其中蛋白质结构和动力学的测量是最有趣的，但也是最具挑战性的。

Intellectual Merit: The border between the interior and exterior of a living cell is a membrane that is only a few nanometers thick. Proteins embedded in cell membranes are the molecular machines that control the flow of matter, energy and information across the border of the cell, and understanding their structures helps to understand how they function. Unfortunately, structural techniques like crystallography and NMR don't work as well for membrane proteins, so new methods are needed. This research project will develop new molecular probes and experimental methods that will provide information on protein structure. The new probes take advantage of the remarkable magnetic properties of ions of the element gadolinium (Gd3+), which are already used to enhance contrast in magnetic resonance imaging (MRI). When excited by a laser inside a very high magnetic field, gadolinium ions precess (wobble) like tops at a frequency of 240 billion cycles per second (240 gigahertz); this occurs about 20,000 times before relaxing most of the way to thermal equilibrium. When a Gd3+ ion is close to another, it relaxes more quickly. Moreover, when the two are less than 10 nanometers apart, the precession amplitude oscillates between the two ions at a "beat frequency" that varies sensitively with the distance between the ions. Together, the relaxation times, precession frequencies, and beat frequency give distance (and ultimately structural) information. These parameters will be measured using newly developed electron paramagnetic resonance (EPR) spectrometers at UC Santa Barbara (240 gigahertz) and the Weizmann Institute of Science in Israel (95 gigahertz). To calibrate the new methodologies, measurements of the distance between gadolinium ions will be performed on "ruler" compounds consisting of Gd3+ ions separated by a stiff, linear polymer to known distances between 2 and 12 nm. Distance measurements will then be performed between carefully-chosen sites on proteorhodopsin. Proteorhodopsin is a light-activated proton pump isolated from a marine bacterium and is similar in structure to membrane proteins that occur in all other organisms. Broader impacts: The project is a new interdisciplinary international collaboration between the U. S. (Song-I Han and Mark Sherwin, UC Santa Barbara), Israel (Daniella Goldfarb, Weizmann Institute of Science), and Germany (Adelheidt Godt, University of Bielefeld, probe synthesis). The collaborators and co-PI are female physicists with considerable expertise in developing new technology. This proposal will support two graduate students and one undergraduate. These students are cross-trained by shuttling between the Han lab (physical chemistry and biochemistry) and the Sherwin lab (experimental physics), and are thus immersed in the science of "soft matter," magnetic resonance, proteins, microwave electronics, terahertz science and technology, and visible optics. Students also will perform experiments in the remarkable magnetic resonance community at the Weizmann Institute of Science in Israel, greatly enriching their training with an international scientific experience. The Gd3+ spin labels and high-field electron paramagnetic resonance techniques that the students develop will enable new studies of membrane proteins in life-like environments by many other scientists. These labels are stable enough to be used inside the complex environment of living cells, where measurements of protein structure and dynamics are most interesting but also most challenging.