Triggered functional dynamics of proteins in biomimetic environments by time-resolved electron paramagnetic resonance at very high magnetic fields

蛋白质是纳米级的分子机器，为我们所知的生命提供动力。多亏了结构生物学的一场革命，我们现在知道了超过15万种蛋白质的三维形状。然而，为了充分理解蛋白质作为机器的运作，人们必须了解它们在被外部事件触发后是如何运动的，比如附着在特定分子上，邻近蛋白质的运动，或者电压或光强度的变化。一类特别令人兴奋的蛋白质被称为光反应蛋白，当暴露在光线下时，它们可以移动来控制细胞的行为。这项研究将开发一种先进的磁共振技术——其中最常见的应用是磁共振成像（MRI）——将光反应蛋白对闪光的反应拍成“电影”。“拍摄”蛋白质运动的能力将提供关键的工具，指导广泛的工程和优化蛋白质的重要应用，从可持续制造到用光控制神经。该项目将通过PI小组开发的“问答板”进行公众宣传活动，吸引下一代科学家。正如我们所知，蛋白质是为生命提供动力的分子机器。这个快速增长的蛋白质数据库现在拥有超过15万个蛋白质结构。然而，为了充分理解蛋白质作为机器的运作，我们必须理解它们触发的功能动力学——在被外部事件（如配体对接、相邻蛋白质的运动或电压或光强度的变化）触发后，蛋白质结构如何及时进化。光响应蛋白是一类特别令人兴奋的蛋白质，它们在吸收光子后产生机械能来调节生化过程和细胞行为。该项目将支持聚合研究，以测量光脉冲在两种光响应蛋白中引发的时间分辨构象变化：一种微生物视紫红质质子泵和光遗传蛋白，称为光，氧和电压（LOV）传感域。测量是由PI和co-PI在过去十年中开发的一种独特的方法实现的，该方法将电子顺磁共振（EPR）在非常高的磁场（8.6特斯拉）和频率（240 GHz）下与基于Gd （III）顺磁性金属中心的化合物的定向诱变和自旋标记相结合。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理集群的支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Proteins are the nano-scopic molecular machines that power life as we know it. Thanks to a revolution in structural biology, we now know the three-dimensional shapes of more than 150,000 proteins. However, to fully understand the operation of proteins as machines, one must understand how they move after being triggered by an external event such as attaching to a particular molecule, movement of a neighboring protein, or a change in voltage or light intensity. A particularly exciting class of proteins called photo responsive proteins can move to control the behaviors of cells when exposed to light. This research will develop an advanced form of magnetic resonance—of which the most familiar application is magnetic resonance imaging (MRI)—to make “movies” of the motion of photo responsive proteins in response to a flash of light. The ability to “film” protein motion will provide critical tools to guide widespread efforts to engineer and optimize proteins for important applications ranging from sustainable manufacturing to controlling nerves with light. This project will engage next generation of scientists by public outreach activities through the “Questboard” developed by the PI’s group. Proteins are the molecular machines that power life as we know it. The rapidly-growing protein data bank now holds more than 150,000 protein structures. However, to fully understand the operation of proteins as machines, one must understand their triggered functional dynamics--how protein structures evolve in time after being triggered by an external event such as ligand docking, movement of a neighboring protein, or a change in voltage or light intensity. Photo responsive proteins are a particularly exciting class of proteins that, after absorbing a photon, generate mechanical energy to modulate biochemical processes and cellular behavior. This project will support convergent research to measure the time-resolved conformational changes triggered by a pulse of light in two photo responsive proteins: a microbial rhodopsin proton pump, and optogenetic proteins called Light, Oxygen, and Voltage (LOV) sensing domains. The measurements are enabled by a unique methodology, under development over the last decade by the PI and co-PI, which combines electron paramagnetic resonance (EPR) at very high magnetic fields (8.6 Tesla) and frequencies (240 GHz) with site-directed mutagenesis and spin labeling using compounds based on Gd (III) paramagnetic metal centers. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.