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

用于膜蛋白结构研究的基于 Gd3 的稳健自旋标签

基本信息

批准号：
1244651
负责人：
Mark Sherwin
金额：
$ 84.85万
依托单位：
University of California-Santa Barbara
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-01 至 2016-12-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1244651&HistoricalAwards=false
关键词：
Robust Gd3+based spin labels

项目摘要

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.

智力优点：活细胞内部和外部之间的边界是只有几纳米厚的膜。嵌入细胞膜中的蛋白质是控制物质，能量和信息在细胞边界的流动的分子机，并且了解它们的结构有助于了解它们的功能。不幸的是，晶体学和NMR等结构技术对于膜蛋白不行，因此需要新的方法。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。新探针利用了元素gadolinium（GD3+）离子的显着磁性，这些特性已经用于增强磁共振成像（MRI）中的对比度。当激光在非常高的磁场内激发时，gadolinium离子螺旋序（Wobble）像上衣一样，每秒2400亿个循环（240 Gigahertz）；在放松大部分方向前达到热平衡之前，这发生了大约20,000次。当GD3+离子接近另一个时，它会更快放松。此外，当两者相距少于10纳米时，进进幅度在两个离子之间以“节拍频率”振荡，这与离子之间的距离敏感。放松时间，预进时间频率和频率共同提供距离（并最终是结构性）信息。这些参数将使用新近开发的电子顺磁共振（EPR）光谱仪（240 Gigahertz）和以色列的Weizmann科学学院（95 Gigahertz）进行测量。为了校准新的方法，将对gadolinium离子之间的距离的测量测量，这些距离将对由GD3+离子组成的“标尺”化合物进行，这些GD3+离子由刚性，线性聚合物分离为2至12 nm之间的已知距离。然后，将在蛋白淡季精心选择的位点之间进行距离测量。蛋白质淡淡蛋白酶是一种从海洋细菌分离的光激活的质子泵，其结构与所有其他生物中发生的膜蛋白相似。更广泛的影响：该项目是美国（Song-I Han和Mark Sherwin，UC Santa Barbara），以色列（以色列）（Daniella Goldfarb，Weizmann科学学院）和德国（Adelheidt Godt，Bielefeld，Bielefeld，Bielefeld，Probe Cynthesis）之间的新的跨学科国际合作。合作者和Co-Pi是在开发新技术方面具有丰富专业知识的女性物理学家。该建议将支持两名研究生和一名本科生。这些学生是通过Han Lab（物理化学和生物化学）和Sherwin Lab（实验物理学）之间的穿梭训练的，因此沉浸在“软物质”，磁共振，蛋白质，蛋白质，微波电子，Terahertz科学和技术以及可见光的科学中。学生还将在以色列魏兹曼科学学院的杰出磁共振社区中进行实验，并通过国际科学经验丰富了他们的培训。学生开发的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在类似生活的环境中对膜蛋白进行新的研究。这些标签足够稳定，可以在活细胞的复杂环境中使用，在这种细胞的复杂环境中，蛋白质结构和动力学的测量最有趣，但也最具挑战性。