Light-Induced Protein Quake of Visual Rhodopsin Investigated by Femtosecond Time-Resolved X-Ray Scattering

从广义上讲，解决的问题是生物信号是如何产生的一类膜蛋白被称为G蛋白偶联受体（GPCR）。 视紫红质是参与视觉的主要GPCR，并且充当用于确定作用中的受体的分子电影的原型。 研究将探讨从视网膜视杆细胞膜提取的视觉视紫红质进入洗涤剂胶束或小脂质纳米盘的光激活机制。为了监测蛋白质的形状和运动，该项目将采用强大的新X射线自由电子激光（XFEL）技术。 目标是研究光如何影响蛋白质的形状和视紫红质在不同时间尺度范围内的内部运动。 其意义和影响是双重的。 首先，它将阐明发生在我们眼睛中的光激活过程的最早事件和物理学，以及产生视觉的蛋白质变化。 其次，它将展示和推动新的XFEL技术在膜蛋白中的应用，这些膜蛋白不适合标准结晶方法。 主要的好处是了解视觉视紫红质的光吸收如何导致其移动性的变化，然后将神经信号传输到大脑。 对洗涤剂溶液中视紫红质的时间分辨X射线研究将揭示由其辅因子（视网膜，维生素A的衍生物）的光吸收引发的蛋白质运动。 计算机模拟将进一步解释实验观察到的蛋白质分子因光而发生的动力学变化。 重要的更广泛的成果包括在博士后，研究生，本科生和高中水平的生物物理科学家的培训，以及教师谁将影响我们的社会在未来许多年。研究的目标和范围涉及时间分辨X射线散射研究洗涤剂溶液和脂质纳米盘中视紫红质的光活化。 由于其光活化作用，视紫红质的结构动力学变化将在多个时间和空间尺度上建立：从亚皮秒（10 C12 s）到毫秒（10 C3 s），从化学键长到整个蛋白质分子。 来自光学参量放大器（OPA）的可见光将被用作泵浦，XFEL的短强X射线脉冲作为探针。 泵-探测研究将建立如何超快的构象变化的视紫红质传播的多尺度机制到受体的激活状态。 辅因子诱导的光视紫红质和视紫红质中间体中蛋白质动力学的变化将从皮秒到纳秒的时间尺度进行探测，以发现初始光吸收后是否立即发生“蛋白质地震”。然后，我们将研究最初的蛋白质震动是如何传播到大规模的构象波动，激活视紫红质。 Lumi和Meta-I状态将在激活Meta-II状态的准备阶段进行研究。我们将发现视网膜生色团的顺式-反式异构化是如何集中在视紫红质的动力学热点上，从而产生激活的构象变化。 量子力学/分子动力学（QM/MM）模拟将比较理论的差异散射分布的实验溶液X射线数据连接光诱导的变化的原子分辨率的结构。 最后，我们将研究G蛋白转导素的C-末端螺旋的结合与结构和动力学改变的关系。 通过研究光诱导的视网膜异构化的超快变化如何与大规模蛋白质波动相结合，将极大地提高我们对视紫红质激活及其在视觉信号中作用的理解。该奖项反映了NSF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。

In broad terms the problem addressed is how biological signals are generated by a class of membrane proteins known as G-protein-coupled receptors (GPCRs). Rhodopsin is the major GPCR involved in vision, and serves as an archetype for determining molecular movies of receptors in action. Research will investigate the light activation mechanism of visual rhodopsin extracted from the retinal rod disk membranes into detergent micelles or small lipid nanodiscs. To monitor the protein shape and motion, the project will apply powerful new X-ray free electron laser (XFEL) technology. The goal is to study how light affects the protein shape and internal motions of rhodopsin over a range of different time scales. The significance and impact are twofold. First, it will illuminate the earliest events and photophysics of light activation process that occur in our eyes, together with the protein changes that yield vision. Second, it will showcase and drive the application of the new XFEL technology to membrane proteins that are not amenable to standard crystallization approaches. The primary benefit is to understand how light absorption by visual rhodopsin leads to changes in its mobility followed by transmission of a nerve signal to the brain. Time-resolved X-ray studies of rhodopsin in detergent solutions will reveal the protein motions triggered by light absorption of its cofactor (retinal, a derivative of Vitamin A). Computer simulations will further interpret the experimental observations in terms of changes in the dynamics of the protein molecules due to light. Important broader outcomes include training of biophysical scientists at the postdoctoral, graduate student, undergraduate, and high school levels, as well as teachers who will influence our society over many years to come. The goals and scope of the research involve time-resolved X-ray scattering studies of the light activation of rhodopsin in detergent solutions and lipid nanodiscs. Changes in the structural dynamics of rhodopsin due to its photoactivation will be established over multiple scales of time and space: from sub picoseconds (10 C12 s) up to milliseconds (10 C3 s), and from chemical bond lengths up to entire protein molecules. Visual light from an optical parametric amplifier (OPA) will be used as the pump, with the short intense X-ray pulses of an XFEL as the probe. Pump-probe studies will establish how the ultrafast conformational changes of rhodopsin are propagated by a multiscale mechanism into the activated state of the receptor. The cofactor-induced changes in protein dynamics in the Photorhodopsin and Bathorhodopsin intermediates will be probed from the picosecond up to the nanosecond time scales to discover whether a "protein quake" occurs immediately after the initial light absorption. We will then study how the initial protein quake is propagated into the large-scale conformational fluctuations that activate rhodopsin. The Lumi and Meta-I states will be studied in the run-up to the active Meta-II state. We will discover how cis Ctrans isomerization of the retinal chromophore is focused to the dynamical hot spots of rhodopsin, which yield the activating conformational changes. Quantum mechanical/molecular dynamics (QM/MM) simulations will compare the theoretical difference-scattering profiles to experimental solution X-ray data to connect the light-induced changes to the atomic-resolution structure. Lastly, we will investigate binding of the C-terminal helix of the G-protein transducin in relation to the structural and dynamical alterations. Establishing how the ultrafast changes from light-induced isomerization of retinal are coupled to the large-scale protein fluctuations will greatly improve our understanding of rhodopsin activation and its role in visual signaling.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.