CAREER: Conformational Changes in Voltage-Controlled Ion Channels Measured by Advanced Fluorescence Techniques

这个项目描述了一个跨学科的五年职业发展计划，在研究和教学中涉及物理、化学和生物。在研究中，将使用两种互补的荧光技术来研究离子通道-负责神经传导的通道-的构象变化。这些技术，一种测量距离，另一种测量旋转，可以应用于许多其他类型的蛋白质和通道。第一种技术是基于镧系元素的共振能量转移(LRET)，它是一种已有技术的改进版本，称为荧光共振能量转移。之所以实现这种改进的分辨率，是因为稀土元素可以通过对两个标记之间距离的变化很敏感的机制将能量转移到有机染料。第二种方法是利用磷光探针或我们最近发现的偏振发光稀土探针来实现磷光各向异性。在该技术中，偏振激发脉冲选择性地激励那些与脉冲偏振相对准的探头，偏振敏感探测器监测探头的重定向。这项技术的优点是它可以测量毫秒级的旋转运动，毫秒级的时间尺度与离子通道中的蛋白质重排有关。LRET将被用来测量特定位置放置的稀土原子和有机染料之间的距离，作为细胞膜上电压的函数，并在同时进行电子测量期间将它们与通道内的运动联系起来。通过磷光各向异性测量适当放置的探针的旋转可以进一步表征蛋白质的运动。LRET的初步研究在电压控制离子通道领域取得了一个里程碑，首次测量了离子通道蛋白质随着电压变化而移动时实际距离的变化。这些结果表明，通道的一部分会旋转，这一假说将得到进一步验证。在教学方面，将开发一门主要面向非科学家的本科课程，题为“科学与公民：当前科学、技术和社会中的争议”。本课程将以支持反对意见的形式进行授课，向学生提供当代科学问题的背景介绍，然后是双方倡导者的阅读材料和广泛的以学生为中心的辩论和写作，以提高学生的批判性、分析和沟通能力。主题包括：弹道导弹防御系统的可行性和可取性、臭氧层空洞和全球变暖等环境问题、食品中的农药、环境中的化学物质、基因工程和克隆动物等健康问题。

This project describes an interdisciplinary five-year career development plan involving physics, chemistry, and biology in both research and teaching.In research, two complementary fluorescence techniques will be used to study conformational changes in ion channels-channels that are responsible for nerve conduction. These techniques, one of which measures distance and the other of which measures rotation, can be applied to many other kinds of proteins and channels. The first technique, lanthanide-based resonance energy transfer (LRET), is an improved version of an established technique called fluorescence resonance energy transfer. This improved resolution is achieved because the lanthanide can transfer energy to an organic dye by a mechanism that is sensitive to angstrom changes in distance between the two labels. The second method is a version of phosphorescence anisotropy utilizing either phosphorescent probes or our recently discovered polarized luminescent lanthanide probes. In this technique, a polarized excitation pulse selectively excites those probes aligned with the pulse polarization and polarization-sensitive detection monitors the reorientation of probes. The technique has the advantage that it can measure rotational motion on the millisecond time scale, the time-scale associated with protein rearrangements in ion channels. LRET will be used to measure distances between site-specifically placed lanthanide atoms and organic dyes as a function of voltage across the cell membrane and relate them to motion within the channel during simultaneous electrical measurements. Measuring rotation of appropriately placed probes by phosphorescence anisotropy can further characterize protein movement. Initial studies with LRET have achieved a milestone in the field of voltage-controlled ion channels, the first measurements of actual distance changes as ion channel proteins move in response to voltage changes. These results suggest that part of the channel rotates, a hypothesis that will be tested further. In teaching, an undergraduate course intended primarily for non-scientists, entitled "Science and the Citizen: Current controversies in science, technology and society" will be developed. The course will be taught in a "pro-con" format with introductory lectures giving students background in contemporary science issues, followed by readings from advocates of both sides and extensive student-centered debate and writing to enhance students' critical, analytical, and communication skills. Topics will include: the feasibility and desirability of a ballistic missile defense system, environmental issues such as the ozone hole and global warming, health issues such as pesticides in food, chemicals in the environment, genetic engineering, and cloning animals.