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对彼此的亲和力。监视 调节蛋白质相互作用为细胞过程提供了重要的机械洞察力，IS 在疾病状态下开发药理学干预至关重要。当前的蛋白质研究 体内和活细胞中的相互作用通常采用荧光共振能量转移（FRET） 在两个遗传编码的荧光蛋白之间提取有关分子间和分子内的信息 蛋白质的变化。但是，这种理解细胞信号传导的方法严重阻碍了两个主要的 障碍：（1）在活细胞中，没有任何方法可以系统地改变局部相互作用的浓度 蛋白质是为了将相互作用转化为细胞功能。 （2）常规的FRET检测严重遭受 由于供体和受体发射发生的很小的变化，从低信噪比（SNR）开始 在蛋白质相互作用中，以及强大的先前供体和受体发射背景。 我们建议开发一个optofluidic fret激光系统，该系统协同两个不同的新出现 技术，系统的蛋白质亲和力强度调节（痉挛）和光氟激光器 以可控制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。 痉挛依靠模块化和可调的ER/K a螺旋来连接两个相互作用的蛋白质。 er/k a-helix可以是 设计为系统地改变蛋白质相互作用频率。同时，Optofluidic激光充当 高度敏感的FRET检测器，可提供定量读数。它采用刺激的激光排放作为 传感信号。当激光腔内发生FRET时，蛋白质引起的FRET发生了少量变化 相互作用将通过optofluidic激光器光学扩增，从而导致FRET急剧增加 信号。此外，由于Optofluidic激光器的独特光学设计，已有的供体和受体 发射背景几乎可以消除。因此，FRET的数量级提高了 可以获得灵敏度。在这里，我们将首先使用ER/K系统地研究OptoFluidic FRET激光器 A-螺旋调制蛋白质fret配对并基准我们针对常规FRET检测的技术。 然后，我们将使用Optofluidic激光研究ER/K A螺旋调节钙调蛋白（CAM） - 肽系统 在体外和活细胞中，其相互作用可以通过Ca2+浓度广泛变化。最后，我们将应用 Optofluidic FRET激光器可以实质上增强（> 100倍）活细胞G蛋白耦合的灵敏度 受体（GPCR）活化传感器使用痉挛技术开发。我们有三个具体的目标： AIM 1：使用ER/K A螺旋调制蛋白质配对进行调查和基准测试和基准测试。 AIM 2：研究并基准用CAM肽内和活细胞中的Optofluidic FRET激光； AIM 3：实质上增强了活细胞GPCR激活传感器的灵敏度（> 100倍）。