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

• 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

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一层只有几纳米厚的膜。 嵌入细胞膜中的蛋白质是控制物质、能量和信息跨细胞边界流动的分子机器，了解它们的结构有助于了解它们的功能。不幸的是，晶体学和核磁共振等结构技术对于膜蛋白来说效果不佳，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，以提供蛋白质结构的信息。新探头利用了钆 (Gd3+) 离子的卓越磁性，该离子已用于增强磁共振成像 (MRI) 的对比度。当在非常高的磁场内被激光激发时，钆离子会像陀螺一样以每秒 2400 亿周期（240 GHz）的频率进动（摆动）；在大部分松弛到热平衡之前，这种情况会发生大约 20,000 次。 当 Gd3+ 离子靠近另一个时，它弛豫得更快。此外，当两者相距小于10纳米时，进动幅度会以“拍频”在两个离子之间振荡，该“拍频”随离子之间的距离而敏感地变化。弛豫时间、进动频率和拍频共同给出距离（以及最终的结构）信息。这些参数将使用加州大学圣巴巴拉分校（240 GHz）和以色列魏茨曼科学研究所（95 GHz）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，将在由 Gd3+ 离子组成的“标尺”化合物上进行钆离子之间的距离测量，这些 Gd3+ 离子被刚性的线性聚合物分隔至 2 至 12 nm 之间的已知距离。然后将在变形视紫红质上仔细选择的位点之间进行距离测量。 蛋白视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体中存在的膜蛋白相似。 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，调查）之间的一项新的跨学科国际合作合成）。 合作者和联合首席研究员是女性物理学家，在开发新技术方面拥有丰富的专业知识。 该提案将支持两名研究生和一名本科生。 这些学生穿梭于汉实验室（物理化学和生物化学）和宣威实验室（实验物理）之间进行交叉训练，从而沉浸在“软物质”、磁共振、蛋白质、微波电子学、太赫兹科学中。和技术以及可见光学。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培训。 学生们开发的 Gd3+ 自旋标记和高场电子顺磁共振技术将使许多其他科学家能够在类似生命的环境中对膜蛋白进行新的研究。 这些标记足够稳定，可以在活细胞的复杂环境中使用，其中蛋白质结构和动力学的测量是最有趣但也是最具挑战性的。

项目成果

Gd 3+ –Gd 3+ distances exceeding 3 nm determined by very high frequency continuous wave electron paramagnetic resonance

由甚高频连续波电子顺磁共振测定的 Gd 3 → Gd 3 距离超过 3 nm

• DOI: 10.1039/c6cp07119h
• 发表时间: 2017-01
• 期刊: Physical Chemistry Chemical Physics
• 影响因子: 3.3
• 作者: Clayton, Jessica A.;Qi, Mian;Godt, Adelheid;Goldfarb, Daniella;Han, Songi;Sherwin, Mark S.
• 通讯作者: Sherwin, Mark S.

Reversal of Paramagnetic Effects by Electron Spin Saturation

电子自旋饱和逆转顺磁效应

• DOI: 10.1021/acs.jpcc.8b00312
• 发表时间: 2018-02
• 期刊: The Journal of Physical Chemistry C
• 作者: Jain, Sheetal K.;Siaw, Ting A.;Equbal, Asif;Wilson, Christopher B.;Kaminker, Ilia;Han, Songi
• 通讯作者: Han, Songi