Protein interaction study In-vitro and in live cells with optofluidic lasers

使用光流控激光器进行体外和活细胞中的蛋白质相互作用研究

• 批准号: 8634300
• 负责人: Xudong Fan
• 金额: $ 22.13万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8634300
• 关键词: Adrenergic Receptor; Affinity; Agonist; Benchmarking; Binding; Biological Models; Biomedical Research; Calmodulin; Cell membrane; Cell physiology; Cells; Detection; Disease; Emerging Technologies; Engineering; Fluorescence; Fluorescence Resonance Energy Transfer; Frequencies; G-Protein-Coupled Receptors; In Vitro; Intervention; Investigation; Lasers; Length; Life; Ligands; Link; Measures; Methods; Monitor; Noise; Optics; Peptides; Pharmacotherapy; Play; Proteins; Receptor Activation; Relative (related person); Research; Role; Sampling Studies; Signal Transduction; Surface; System; Techniques; Technology; Tertiary Protein Structure; Translating; biological research; design; detector; disease diagnosis; drug discovery; fluorophore; insight; protein protein interaction; protein structure; public health relevance; research study; sensor; small molecule

Protein-protein interactions play a central role in cellular signaling. The frequency and strength of protein interactions depend on the local concentrations of two proteins and their affinity for one another. Monitoring and modulating protein interactions provides important mechanistic insight into cellular processes and is essential for developing pharmacological intervention in disease states. Current investigation of protein interactions in-vitro and in live cells commonly employ fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins to extract information about inter- and intramolecular changes in proteins. However, this approach to understand cell signaling is seriously impeded by two major obstacles: (1) Within a live cell, no methods exist to systematically vary the local concentration of interacting proteins in order to translate interaction into cellular function. (2) Conventional FRET detection suffers severely from low signal-to-noise-ratio (SNR) due to the very small changes occurring in donor and acceptor emission during protein interactions, and the strong pre-existing donor and acceptor emission background. We propose to develop an optofluidic FRET laser system that synergizes two distinct, emerging technologies, systematic protein affinity strength modulation (SPASM) and the optofluidic laser, for unprecedented capability of analyzing protein interactions in-vitro and in live cells in a controllable manner. SPASM relies on a modular and tunable ER/K a-helix to link two interacting proteins. The ER/K a-helix can be engineered to systematically change the protein interaction frequency. Meanwhile, the optofluidic laser acts as a highly sensitive FRET detector to provide a quantitative readout. It employs stimulated laser emission as the sensing signal. When FRET takes place inside the laser cavity, a small change in FRET induced by protein interactions will be optically amplified by the optofluidic laser, thus resulting in a drastic increase in the FRET signal. In addition, due to the unique optical design of the optofluidic laser, the pre-existing donor and acceptor emission background can virtually be eliminated. Therefore, orders of magnitude improvement in FRET sensitivity can be obtained. Here, we will first systematically investigate the optofluidic FRET laser using ER/K a-helix modulated protein FRET pairs and benchmark our technology against conventional FRET detection. Then, we will use the optofluidic laser to study the ER/K a-helix modulated calmodulin (CAM)-peptide system in-vitro and in live cells, whose interaction can be varied widely by Ca2+ concentration. Finally, we will apply the optofluidic FRET laser to substantially enhance (>100 fold) the sensitivity of a live cell G-protein coupled receptor (GPCR) activation sensor developed using the SPASM technique. We have three specific aims: Aim 1: Investigate and benchmark the optofluidic FRET laser with ER/K a-helix modulated protein FRET pairs; Aim 2: Investigate and benchmark the optofluidic FRET laser with CAM-peptide in-vitro and in live cells; Aim 3: Substantially enhance (>100 fold) the sensitivity of a live cell GPCR activation sensor.

蛋白质-蛋白质相互作用在细胞信号传导中起着核心作用。蛋白质的频率和强度 相互作用取决于两种蛋白质的局部浓度及其相互之间的亲和力。监测 和调节蛋白质相互作用提供了重要的机制洞察细胞过程， 对于发展疾病状态下的药物干预至关重要。蛋白质研究现状 体外和活细胞中的相互作用通常采用荧光共振能量转移（FRET）， 在两个基因编码的荧光蛋白之间提取关于分子间和分子内的信息， 蛋白质的变化。然而，这种理解细胞信号传导的方法受到两个主要因素的严重阻碍。 障碍：（1）在活细胞内，不存在系统地改变相互作用的局部浓度的方法。 蛋白质，以便将相互作用转化为细胞功能。(2)传统的FRET检测受到严重影响， 由于供体和受体发射中发生的非常小的变化， 在蛋白质相互作用期间，以及强的预先存在的供体和受体发射背景。 我们建议开发一种光流FRET激光系统，它可以协同两种不同的，新兴的 技术，系统蛋白质亲和强度调制（SPASM）和光流体激光， 以可控的方式在体外和活细胞中分析蛋白质相互作用的前所未有的能力。 SPASM依赖于模块化和可调的ER/K α-螺旋连接两个相互作用的蛋白质。ER/Ka-螺旋可以是 被设计成系统地改变蛋白质相互作用的频率。同时，光流控激光器作为 高灵敏度FRET检测器，以提供定量读数。它采用受激激光发射作为 传感信号当FRET发生在激光腔内时，蛋白质诱导的FRET的微小变化 相互作用将被光流体激光器光学放大，从而导致FRET的急剧增加 信号了此外，由于光流体激光器的独特光学设计，预先存在的供体和受体 实际上可以消除发射背景。因此，FRET的数量级改善 可以获得灵敏度。在这里，我们将首先系统地研究使用ER/K的光流FRET激光器 α-螺旋调制蛋白FRET对和基准我们的技术对传统的FRET检测。 然后，我们将利用光流控激光器研究ER/Ka-螺旋调控的钙调素（CAM）-肽系统 在体外和活细胞中，其相互作用可以通过Ca 2+浓度广泛变化。最后，我们将应用 光流体FRET激光器，以显著提高（>100倍）的灵敏度的活细胞G-蛋白偶联 受体（GPCR）激活传感器使用SPASM技术开发。我们有三个具体目标： 目的1：研究ER/K α-螺旋调制蛋白质FRET对的光流体FRET激光器并进行基准测试; 目的2：研究和基准的光流体FRET激光与CAM-肽在体外和活细胞; 目的3：显著增强（>100倍）活细胞GPCR活化传感器的灵敏度。