Optical Control of Protein Activity in Live Cells by Plasmon Assisted Light Inactivation

摘要 光学工具具有无与伦比的空间和时间精度，有助于更好地理解 现代医学和生物学中的各种过程。我的研究实验室的总体目标是 了解激光-等离子体纳米粒子相互作用及其在生物界面上的影响 系统和纳米材料。具体地说，实验技术和方法已经发展到 了解纳米粒子等离子体加热对蛋白质和血脂的影响 纳米颗粒。这导致了用于光学蛋白质操纵和光敏的新的使能工具 用于分子去势的纳米微囊，以及创新的诊断方法。这一建议的研究重点 关于活细胞中蛋白质活性的光学控制，即等离子体辅助光灭活的研究进展 (巴利语)。PALi是基于脉冲激光(纳秒)对等离子体纳米粒子的加热，其热 有限加热以在纳米颗粒表面几纳米范围内展开和变性周围的蛋白质。 因此，巴利还有效地发挥了独特的纳米温度跳跃(T-JUMP)这一创新实验平台的作用 以解决蛋白质展开研究方面的空白。在接下来的五年里，我计划发展我的研究 在这两个方向上规划。首先，我将专注于开发这种新的光学工具来操纵蛋白质 活细胞中的活性，重点是G蛋白偶联受体(GPCR)，这是一类重要的和多样化的 介导细胞外信号到细胞内信号的膜受体。这包括系统化的 了解纳米颗粒与GPCR的相互作用和转运的方法，细胞反应 PALI在GPCR信号转导上的应用，以及PALi在其他GPCR上的适用性。我将主要使用一个特定的 在慢性疼痛中起重要作用的蛋白水解酶激活受体2(PAR2)是一种工作模型。测试，测试 其他GPCRs，我将测试GPCRs中的神经肽，这些神经肽与我们创造神经肽的努力是协同的 光敏纳米微囊。其次，我将集中讨论纳米T跳跃的特征 解决两个基本问题：(1)纳米颗粒温度能否在 脉冲激光加热还是在短暂延迟后？(2)蛋白质在纳米T跳跃下是如何展开的？这些 涉及我们与阿贡国家实验室的现有合作，以探测金晶格膨胀 先进的光谱学，以及各种结构和功能分析来测量蛋白质的展开和 由于纳米T跳跃而失活。到五年结束时，我预计将解决重要的技术问题 演示使用PALi通过GPCRs操纵活细胞中的蛋白质活性的挑战，以及 通过创新的纳米T-JUMP清楚地了解温度历史和蛋白质响应 站台。这些成果将引起广泛的研究界的兴趣，并使其他人能够 使用PALI解决细胞生物学中的重要挑战。

Abstract Optical tools have unparalleled spatial and temporal precision and have been instrumental to better understand various processes in modern medicine and biology. The overall goal of my research laboratory is to understand the laser-plasmonic nanoparticle interactions and its effects at the interface between biological systems and nanomaterials. Specifically, experimental techniques and methods have been developed to understand the effects of nanoparticle plasmonic heating on proteins and lipids immediately next to the nanoparticle. This has led to new enabling tools for optical protein manipulation and photosensitive nanovesicles for molecular uncaging, as well as innovative diagnostic methods. This proposed research focus on the development of optical control of protein activity in live cells, namely plasmon-assisted light inactivation (PALI). PALI is based on pulsed laser heating (nanosecond) of plasmonic nanoparticles, and its thermally confined heating to unfold and denature surrounding proteins within a few nanometers of nanoparticle surface. Thus, PALI also effectively acts a unique nano temperature-jump (T-jump), an innovative experimental platform to address a gap for protein unfolding investigations. In the next five years, I plan to develop my research program in these two directions. Firstly, I will focus on developing this new optical tool to manipulate protein activity in live cells with emphasis on G-protein coupled receptors (GPCR), an important and diverse class of membrane receptors that mediate extracellular to intracellular signaling. This encompasses a systematic approach to understand the interaction and trafficking of nanoparticles with GPCR, the cellular responses of PALI on GPCR signaling, and finally the applicability of PALI on other GPCRs. I will primarily use a specific GPCR, protease activated receptor 2 (PAR2) that is important for chronic pain, as a working model. To test for other GPCRs, I will test GPCRs for neuropeptides, which are synergistic with our efforts to create neuropeptide photosensitive nanovesicles. Secondly, I will concentrate on the characterization of the nano T-jump by addressing two fundamental questions: (1) can the nanoparticle temperature be directly measured during pulsed laser heating or after a short delay? (2) How does the protein unfold under nano T-jump? These involves our existing collaborations with the Argonne National Lab to probe the gold lattice expansion using advanced spectroscopy, and various structural and functional assays to measure the protein unfolding and inactivation due to the nano T-jump. By the end of the five years, I anticipate solving important technical challenges to demonstrate the use of PALI to manipulate protein activity in live cells through GPCRs, and obtain a clear understanding of the temperature history and protein responses with the innovative nano T-jump platform. These outcomes would generate interest to the broad research community and enable others to tackle important challenges in cell biology using PALI.