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

通过等离激元辅助光灭活对活细胞中蛋白质活性的光学控制

• 批准号: 10223375
• 负责人: Zhenpeng Qin
• 金额: $ 38.25万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223375
• 关键词: Address; Biological Assay; Biology; Cells; Cellular biology; Collaborations; Communities; Development; Diagnostic Procedure; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTPase-Activating Proteins; Goals; Gold; Heating; Investigation; Laboratory Research; Lasers; Light; Lipids; Location; Measures; Mediating; Membrane; Methods; Modeling; Modern Medicine; Molecular; Neuropeptide Receptor; Neuropeptides; Optical Methods; Optics; Outcome; PAR-2 Receptor; Photosensitivity; Physiologic pulse; Process; Protein Conformation; Proteins; Recording of previous events; Research; Signal Transduction; Spectrum Analysis; Structure; Surface; Techniques; Temperature; Testing; Time; Work; base; biological systems; chronic pain; extracellular; innovation; insight; interest; nano; nanomaterials; nanometer; nanoparticle; nanosecond; nanovesicle; novel; plasmonics; programs; receptor; response; success; temperature jump; tool; trafficking

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.

抽象的 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-等离子体纳米颗粒相互作用及其在生物之间界面的影响 系统和纳米材料。具体来说，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紧邻的蛋白质和脂质的影响 纳米粒子。这催生了用于光学蛋白质操纵和光敏的新工具。 用于分子解笼的纳米囊泡以及创新的诊断方法。本次拟定的研究重点 活细胞中蛋白质活性光学控制的发展，即等离子体辅助光灭活 （巴利语）。 PALI 基于等离子体纳米颗粒的脉冲激光加热（纳秒），其热学特性 有限的加热使纳米颗粒表面几纳米内的周围蛋白质展开和变性。 因此，PALI还有效地发挥了独特的纳米温度跳跃（T-jump）创新实验平台的作用 解决蛋白质展开研究的空白。未来五年，我计划发展我的研究 规划在这两个方向。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)，这是一类重要且多样化的受体 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 方法来了解纳米颗粒与 GPCR 的相互作用和运输、细胞反应 PALI 对 GPCR 信号传导的影响，最后是 PALI 对其他 GPCR 的适用性。我将主要使用特定的 GPCR，蛋白酶激活受体 2 (PAR2)，作为工作模型对慢性疼痛很重要。测试 其他 GPCR，我将测试神经肽的 GPCR，这与我们创造神经肽的努力具有协同作用 光敏纳米囊泡。其次，我将重点关注纳米 T 跳跃的表征 解决两个基本问题：（1）可以直接测量纳米颗粒的温度吗？ 脉冲激光加热还是短暂延迟后加热？ （2）纳米T跳跃下蛋白质如何展开？这些 涉及我们与阿贡国家实验室现有的合作，利用 先进的光谱学以及各种结构和功能测定来测量蛋白质的解折叠和 由于纳米 T 跳跃而失活。到五年结束时，我预计解决重要的技术问题 挑战证明使用 PALI 通过 GPCR 操纵活细胞中的蛋白质活性，以及 通过创新的纳米 T-jump 清晰地了解温度历史和蛋白质反应 平台。这些成果将引起广泛研究界的兴趣，并使其他人能够 使用 PALI 解决细胞生物学中的重要挑战。