New probes for optical switching of protein interactions and protein fluorescence

用于蛋白质相互作用和蛋白质荧光光学开关的新探针

• 批准号: 7938234
• 负责人: GERARD MARRIOTT
• 金额: $ 40.96万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7938234
• 关键词: Achievement; Actins; Albumins; Binding Proteins; Cell physiology; Cells; Complex; Confocal Microscopy; Coupled; Cytoskeleton; Development; Event; Extinction (Psychology); Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Frequencies; Goals; Image; Image Analysis; Imaging Techniques; In Vitro; Life; Maps; Measures; Mediating; Microfilaments; Microscope; Microscopy; Molecular; Optics; Pan Genus; Phosphatidylinositol 4,5-Diphosphate; Physiology; Platelet-Derived Growth Factor Receptor; Plus End of the Actin Filament; Process; Property; Proteins; Publications; Quantitative Microscopy; Receptor Signaling; Regulation; Research; Research Personnel; Resolution; Role; Signal Transduction; Signaling Molecule; Signaling Protein; Site; Sulfhydryl Compounds; Techniques; Technology; Testing; Variant; Yin; design; fluorescence imaging; in vivo; insight; meetings; membrane activity; optical switch; polymerization; programs; protein protein interaction; receptor; receptor-mediated signaling; response

DESCRIPTION (provided by applicant): New probes for optical switching of protein interactions and protein fluorescence. Cell protrusion is characterized by spatial and temporal fluctuations in the concentrations, interactions and activities of membrane receptors, signaling proteins and 2nd messengers that include PDGF receptor, Ca2+, PIP2/PIP3, CapG and the barbed end of the actin filament. These molecular events are coupled to the rapid polymerization of actin filaments at sites close to the activated receptor. Efforts to elucidate the mechanism of cell protrusion are made difficult, in part, because of the paucity of probes to map specific interactions at the barbed end, and the need for specialized techniques to rapidly map, manipulate and measure the interactions and activities of specific signaling molecules and their target proteins in live cells. We describe a technology/hypothesis-driven approach to meet the research objective that introduces new optical probes including optical switches and imaging techniques that will be used to establish how molecular signals generated during receptor-mediated signaling are coupled to the spatial and temporal regulation of the CapG-barbed end complex, actin polymerization and cell protrusion. The proposal has four specific aims: Aim 1: To design and characterize new reactive optical switches and their conjugates. Aim 2: To design, synthesize and characterize optical switches to reversibly modulate cell Ca2+. Aim 3: To understand how a change in cell Ca2+ is coupled to the regulation of free barbed ends. Aim 4: To establish the role of PIP2/PIP3- dependent uncapping of barbed-ends during cell protrusion. The feasibility of the proposed studies is demonstrated through key preliminary achievements and recent publications. We believe that the unique properties of the new optical switches and associated technologies introduced in this proposal will spur the development of new microscope techniques and advance mechanism-driven studies of complex and dynamic processes in cell and molecular physiology.

描述（由申请人提供）：用于蛋白质相互作用和蛋白质荧光的光学开关的新探针。细胞突起的特征在于膜受体、信号蛋白和第二信使（包括PDGF受体、Ca 2+、PIP 2/PIP 3、CapG和肌动蛋白丝的倒刺末端）的浓度、相互作用和活性的空间和时间波动。这些分子事件与靠近激活受体的肌动蛋白丝的快速聚合有关。努力阐明细胞突起的机制是困难的，部分原因是缺乏探针来绘制倒刺末端的特异性相互作用，以及需要专门的技术来快速绘制、操纵和测量活细胞中特异性信号分子及其靶蛋白的相互作用和活性。我们描述了一种技术/假设驱动的方法，以满足研究目标，引入新的光学探针，包括光学开关和成像技术，将用于建立如何在受体介导的信号传导过程中产生的分子信号耦合到空间和时间调节的CapG-倒刺末端复合物，肌动蛋白聚合和细胞突起。该提案有四个具体目标：目标1：设计和表征新的反应式光开关及其共轭物。目的2：设计、合成和表征可逆调控细胞内钙离子的光开关。目的3：了解细胞Ca 2+的变化如何与自由倒刺末端的调节相结合。目的4：建立PIP 2/PIP 3依赖的倒钩末端去帽化在细胞突起过程中的作用。通过主要的初步成果和最近的出版物证明了拟议研究的可行性。我们相信，本提案中介绍的新型光开关和相关技术的独特性能将推动新的显微镜技术的发展，并推进细胞和分子生理学中复杂和动态过程的机制驱动研究。

项目成果

Probing conformational changes of prestin with thiol-reactive optical switches.

用硫醇反应光开关探测 prestin 的构象变化。

• DOI: 10.1529/biophysj.108.132878
• 发表时间: 2008
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Fang,Jie;Sakata,Tomoyo;Marriott,Gerard;Iwasa,KuniH
• 通讯作者: Iwasa,KuniH

Structural and biochemical studies of actin in complex with synthetic macrolide tail analogues.

• DOI: 10.1002/cmdc.201402150
• 发表时间: 2014-10
• 期刊: CHEMMEDCHEM
• 影响因子: 3.4
• 作者: Pereira, Jose H.;Petchprayoon, Chutima;Hoepker, Alexander C.;Moriarty, Nigel W.;Fink, Sarah J.;Cecere, Giuseppe;Paterson, Ian;Adams, Paul D.;Marriott, Gerard
• 通讯作者: Marriott, Gerard