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

从广义上讲，研究的问题是一类被称为g蛋白偶联受体（gpcr）的膜蛋白如何产生生物信号。视紫红质是参与视觉的主要GPCR，并作为确定受体分子电影的原型。研究将探讨视紫红质从视网膜棒盘膜中提取成洗涤剂胶束或小脂质纳米盘的光活化机制。为了监测蛋白质的形状和运动，该项目将应用强大的新型x射线自由电子激光（XFEL）技术。目的是研究光如何在不同的时间尺度上影响蛋白质的形状和视紫红质的内部运动。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最早的光激活过程和光物理学，以及产生视力的蛋白质变化。其次，它将展示并推动新的XFEL技术在膜蛋白上的应用，这些膜蛋白不适合标准的结晶方法。主要的好处是了解视紫红质的光吸收如何导致其移动性的变化，然后将神经信号传递到大脑。洗涤剂溶液中紫红质的时间分辨x射线研究将揭示由其辅助因子（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由于光引起的蛋白质分子动力学变化方面的实验观察结果。重要的更广泛的成果包括培养博士后、研究生、本科生和高中水平的生物物理科学家，以及将在未来许多年影响我们社会的教师。该研究的目标和范围包括对洗涤剂溶液和脂质纳米圆盘中紫红质光激活的时间分辨x射线散射研究。视紫红质因其光激活而引起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），从化学键长度到整个蛋白质分子。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射线脉冲作为探头。泵-探针研究将确定视紫红质的超快构象变化是如何通过多尺度机制传播到受体激活状态的。在从皮秒到纳秒的时间尺度上，将探索光紫红质和深紫红质中间体中辅因子诱导的蛋白质动力学变化，以发现在初始光吸收后是否立即发生“蛋白质地震”。然后，我们将研究如何将最初的蛋白质震动传播到激活视紫红质的大规模构象波动中。Lumi和Meta-I状态将在进入活跃的Meta-II状态之前进行研究。我们将发现视网膜发色团的顺式Ctrans异构化如何聚焦于视紫质的动态热点，从而产生激活的构象变化。量子力学/分子动力学（QM/MM）模拟将比较理论差异散射曲线与实验溶液x射线数据，以将光诱导的变化与原子分辨率结构联系起来。最后，我们将研究g蛋白转导蛋白c端螺旋的结合与结构和动力学改变的关系。建立视网膜光诱导异构化的超快变化如何耦合到大规模的蛋白质波动将大大提高我们对视紫红质活化及其在视觉信号传导中的作用的理解。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。

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.