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， 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的相互作用及其在生物之间的界面上的影响 系统和纳米材料。具体而言，已经开发了实验技术和方法， 了解纳米粒子等离子体加热对蛋白质和脂质的影响， 纳米粒子。这导致了光学蛋白质操纵和光敏化的新工具 纳米囊泡的分子解开，以及创新的诊断方法。本研究重点 关于活细胞中蛋白质活性的光学控制的发展，即等离子体辅助的光失活 （帕利）。帕利基于等离子体纳米粒子的脉冲激光加热（纳秒），并且其热 限制加热以使纳米颗粒表面的几纳米内的周围蛋白质展开和变性。 因此，帕利还有效地充当了独特的纳米温度跳跃（T-jump），一个创新的实验平台 以填补蛋白质解折叠研究的空白。在接下来的五年里，我计划发展我的研究 这两个方向的计划。首先，我将致力于开发这种新的光学工具来操纵蛋白质 在活细胞中的活性，重点是G蛋白偶联受体（GPCR），一种重要而多样的 介导细胞外到细胞内信号传导的膜受体。这包括一个系统的 一种了解纳米颗粒与GPCR的相互作用和运输的方法， PALI对GPCR信号传导的影响，以及帕利对其他GPCR的适用性。我将主要使用一个特定的 GPCR，蛋白酶激活受体2（PAR 2），这是重要的慢性疼痛，作为一个工作模型。以测试 其他GPCR，我将测试GPCR中的神经肽，这与我们创造神经肽的努力是协同的。 光敏纳米囊泡。其次，我将集中在表征纳米T-跳跃， 解决了两个基本问题：（1）纳米颗粒的温度是否可以直接测量， 脉冲激光加热或短暂延迟后？(2)蛋白质如何在纳米T跳下展开？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测定，以测量蛋白质解折叠， 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， 证明使用帕利通过GPCR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型跳跃，清晰了解温度历史和蛋白质反应 平台这些成果将引起广大研究界的兴趣，并使其他人能够 使用帕利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.