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

通过甚高频 Gd3 EPR 实现蛋白质的时间分辨构象变化

基本信息

批准号：
1617025
负责人：
Mark Sherwin
金额：
$ 80万
依托单位：
University of California-Santa Barbara
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-15 至 2020-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1617025&HistoricalAwards=false
关键词：
Time resolved conformational changes proteins

项目摘要

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.

该项目旨在开发一种“拍摄”蛋白质的方法，蛋白质是一种微型机器，在执行其关键的生物功能时使我们所知的所有生命得以实现。了解蛋白质的功能是最令人兴奋的科学前沿之一，对于满足可再生能源和农业等不同领域的社会需求是必要的。由于 X 射线光谱、磁共振和其他方法的革命性进步，现已记录了超过 100,000 种不同蛋白质的丰富而复杂的 3 维形状。然而，虽然了解机器的形状（例如，查看缝纫机的照片）可能会给人们提供有关其工作原理的线索，但一部机器运转的电影更具启发性。该项目的目标是开发新方法来制作此类蛋白质“电影”，或者更准确地说，开发测量蛋白质结构（或结构动力学）中时间分辨构象变化的方法。一个由研究生和本科生组成的团队，在物理、化学和生物学之间进行研究，并与国际合作者密切合作，将解决这个令人兴奋的问题，并将成为国家科学、技术、工程领域的领导者“拍摄”活动中的蛋白质需要一个校准工具来测量几纳米的距离，并具有高通量和理想的亚毫秒时间分辨率。该工具必须与蛋白质发挥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高于约 215K 就会继续发挥作用。该研究的主要目标是利用高频 (200 GHz) 电子顺磁共振 (EPR) 与自旋 7/2 Gd3+ 部分的定点自旋标记相结合来测量蛋白质的时间分辨构象变化。在 NSF 先前的支持下，PI 和联合 PI 的合作证明了最简单的 240 GHz EPR 测量；测量含有一对 Gd3+ 自旋标记的分子的线形；即使在室温下也能够分辨大于 3 nm 的距离。拟议的研究将建立在这些关键进展的基础上，主要关注模型蛋白Proteorhodopsin（PR）的时间分辨距离测量，Proteorhodopsin是一种光合跨膜质子泵，可以用闪光触发，开始同步级联构象变化。时间尺度范围从小于 1 微秒到大于 1 秒。所开展的主要活动将是： (1) 开发一种基于测量 Gd3+ EPR 线的线形的简单方法，以从 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) 以亚毫秒时间分辨率测量 PR 在约 215K（其中 PR 冻结但活跃）到室温的温度范围内的构象变化。 (3) Gd3+ 的脉冲 EPR 研究，以了解 Gd3+ 在极高频率下的自旋物理特性。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Quantitative analysis of zero-field splitting parameter distributions in Gd( iii ) complexes

Gd( iii )配合物零场分裂参数分布的定量分析

DOI：
10.1039/c7cp08507a
发表时间：
2018-04-18
期刊：
Physical Chemistry Chemical Physics
影响因子：
3.3
作者：
Clayton JA ;Keller K ;Qi M ;Wegner J ;Koch V ;Hintz H ;Godt A ;Han S ;Jeschke G ;Sherwin MS ;Yulikov M
通讯作者：
Yulikov M

Effect of water/glycerol polymorphism on dynamic nuclear polarization

水/甘油多态性对动态核极化的影响

DOI：
10.1039/c8cp00358k
发表时间：
2018-01
期刊：
Physical Chemistry Chemical Physics
影响因子：
3.3
作者：
Leavesley, Alisa;Wilson, Christopher B.;Sherwin, Mark;Han, Songi
通讯作者：
Han, Songi

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：
2017-02-15
期刊：
Physical Chemistry Chemical Physics
影响因子：
3.3
作者：
Clayton JA;Qi M;Godt A;Goldfarb D;Han S;Sherwin MS
通讯作者：
Sherwin MS

Small Gd(III) Tags for Gd(III)–Gd(III) Distance Measurements in Proteins by EPR Spectroscopy

小 Gd(III) 标签，用于通过 EPR 光谱法测量蛋白质中的 Gd(III)–Gd(III) 距离

DOI：
10.1021/acs.inorgchem.8b00133
发表时间：
2018-04
期刊：
Inorganic Chemistry
影响因子：
4.6
作者：
Prokopiou, Georgia;Lee, Michael D.;Collauto, Alberto;Abdelkader, Elwy H.;Bahrenberg, Thorsten;Feintuch, Akiva;Ramirez;Clayton, Jessica;Swarbrick, James D.;Graham, Bim;et al
通讯作者：
et al

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
影响因子：
0
作者：
Jain, Sheetal K.;Siaw, Ting A.;Equbal, Asif;Wilson, Christopher B.;Kaminker, Ilia;Han, Songi
通讯作者：
Han, Songi