MRI: Development of a Multimodal Instrument for Simultaneous Mechanical and Fluorescence Spectroscopy Measurements of Single Molecules and Molecular Aggregates

MRI：开发用于同时机械和荧光光谱测量单分子和分子聚集体的多模态仪器

• 批准号: 1919670
• 负责人: Ionel Popa
• 金额: $ 98.37万
• 依托单位: University of Wisconsin-Milwaukee
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1919670&HistoricalAwards=false
• 关键词: MRI; Development; Multimodal; Instrument; Simultaneous

An award is made to the University of Wisconsin-Milwaukee (UWM) to support the development of multimodal instrument for simultaneous mechanical and fluorescence spectroscopy measurements of single molecules and molecular aggregates. This instrument will enable cutting-edge research and education in the areas of molecular and cellular biology, high-throughput drug screening and cellular analysis, and development of new biomaterials. This new instrument will be part of the Biophysical Microspectroscopy Facility and will serve several laboratories at UWM, as well as other institutions locally, nationally and abroad. Building on their strong record of involving underrepresented minorities in STEM and training graduate, undergraduate and high school students in their research programs, the PIs will use this project to also educate the next generation of scientists and engineers. During the development phase, undergraduate, graduate and postdoctoral researchers will be involved in the design, custom-manufacturing, computer programming, and development of new protocols for imaging, single molecule spectroscopy and for data analysis. This project will also involve middle school and high school students from the Milwaukee area, and will be used for demonstration purposes and for pilot data acquisition and analysis, respectively. Once completed, this facility will provide novel capabilities to the research programs of numerous early-career and senior investigators; it will also play an essential role in preparing the technologically literate workforce of the future, through elaborate outreach programs, as well as teaching and training of undergraduate, graduate, and post-graduate students that will run during the award period and beyond. The new instrument design will be made available to other researchers or US companies interested in bringing this technology to market.The instrument developed here combines magnetic tweezers, a technique developed by the PI to trap paramagnetic beads and apply mechanical forces to single molecules and cells with Forster resonance energy transfer (FRET) and fluorescence spectroscopy, a method developed by the co-PI to study interactions between molecules and aggregates with spectral resolution. Such a hybrid instrument is not currently commercially available and will require several labor-intensive steps, such as design and fabrication of custom parts, optical alignment and optimization, an integrated control software interface, etc. This instrument presents some unique features. The parallel implementation of force and fluorescence spectroscopy will allow measurements of single-molecule fluorescence with spectral resolution of about 3 nm (over most of the visible spectrum) under mechanical perturbation with a force resolution of 0.1 pN up in the nN range and over hour-to-days sampling times. The multidisciplinary team assembled by the PI is ideally suited to develop and validate this technology, and combines exquisite technical expertise, facilities, experience with developing and running an imaging facility, and close collaborations between physicists, biologists and other life scientists. The instrument will be used for a variety of research studies that involve lateral interactions between proteins under controlled static or dynamic force situations and freely diffusing molecules of a different or the same type, e.g., to study force-induced ligand binding, folding intermediates, antibody detection at single molecule level, receptor-receptor association, and receptor-ligand interactions. The project is jointly funded by the Office of Integrative Activities Major Research Instrumentation Program and the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

威斯康星大学密尔沃基分校 (UWM) 获得了一项奖项，以支持开发多模式仪器，用于同时测量单分子和分子聚集体的机械和荧光光谱。该仪器将促进分子和细胞生物学、高通量药物筛选和细胞分析以及新生物材料开发等领域的前沿研究和教育。该新仪器将成为生物物理显微光谱设施的一部分，并将为 UWM 的多个实验室以及本地、国内和国外的其他机构提供服务。 PI 在让少数族裔参与 STEM 以及在研究项目中培训研究生、本科生和高中生方面拥有良好的记录，在此基础上，PI 将利用该项目来教育下一代科学家和工程师。在开发阶段，本科生、研究生和博士后研究人员将参与设计、定制制造、计算机编程以及成像、单分子光谱和数据分析新协议的开发。 该项目还将涉及密尔沃基地区的中学生和高中生，并将分别用于演示目的以及试点数据采集和分析。一旦建成，该设施将为众多早期职业和高级研究人员的研究项目提供新颖的能力；它还将通过精心设计的外展计划以及在获奖期间及之后对本科生、研究生和研究生的教学和培训，在培养未来具有技术素养的劳动力方面发挥重要作用。新的仪器设计将提供给有兴趣将该技术推向市场的其他研究人员或美国公司。这里开发的仪器结合了磁性镊子，这是由 PI 开发的一种技术，用于捕获顺磁珠，并通过福斯特共振能量转移 (FRET) 和荧光光谱法对单分子和细胞施加机械力，荧光光谱是由共同 PI 开发的一种方法，用于以光谱分辨率研究分子和聚集体之间的相互作用。这种混合仪器目前尚未商用，并且需要几个劳动密集型步骤，例如定制零件的设计和制造、光学对准和优化、集成控制软件界面等。该仪器具有一些独特的功能。力和荧光光谱的并行实施将允许在机械扰动下测量单分子荧光，光谱分辨率约为 3 nm（覆盖大部分可见光谱），力分辨率在 nN 范围内为 0.1 pN，采样时间为数小时至数天。由 PI 组建的多学科团队非常适合开发和验证这项技术，并结合了精湛的技术专业知识、设施、开发和运行成像设施的经验，以及物理学家、生物学家和其他生命科学家之间的密切合作。该仪器将用于各种研究，涉及受控静态或动态力情况下蛋白质与不同或相同类型的自由扩散分子之间的横向相互作用，例如，研究力诱导的配体结合、折叠中间体、单分子水平的抗体检测、受体-受体关联以及受体-配体相互作用。 该项目由综合活动主要研究仪器计划办公室和分子和细胞生物科学部的分子生物物理学集群共同资助。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions

• DOI: 10.1016/j.saa.2021.120133
• 发表时间: 2021-07-07
• 期刊: SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
• 影响因子: 4.4
• 作者: Adhikari, Dhruba P.;Biener, Gabriel;Raicu, Valerica
• 通讯作者: Raicu, Valerica

Proposal for simultaneous analysis of fluorescence intensity fluctuations and resonance energy transfer (IFRET) measurements

• DOI: 10.1088/2050-6120/ab9b68
• 发表时间: 2020-06
• 期刊: Methods and Applications in Fluorescence
• 影响因子: 3.2
• 作者: M. Stoneman;G. Biener;V. Raicu

The efficacy of receptor tyrosine kinase EphA2 autophosphorylation increases with EphA2 oligomer size.

• DOI: 10.1016/j.jbc.2022.102370
• 发表时间: 2022-10
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Zapata-Mercado, Elmer;Biener, Gabriel;McKenzie, Daniel M.;Wimley, William C.;Pasquale, Elena B.;Raicu, Valerica;Hristova, Kalina
• 通讯作者: Hristova, Kalina

In-Cell Detection of Conformational Substates of a G Protein-Coupled Receptor Quaternary Structure: Modulation of Substate Probability by Cognate Ligand Binding

G 蛋白偶联受体四级结构构象亚态的细胞内检测：通过同源配体结合调节亚态概率

• DOI: 10.1021/acs.jpcb.0c06081
• 发表时间: 2020
• 期刊: The Journal of Physical Chemistry B
• 作者: Paprocki, Joel;Biener, Gabriel;Stoneman, Michael;Raicu, Valerică
• 通讯作者: Raicu, Valerică

Cation-induced shape programming and morphing in protein-based hydrogels

• DOI: 10.1126/sciadv.aba6112
• 发表时间: 2020-04-01
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Khoury, Luai R.;Slawinski, Marina;Popa, Ionel
• 通讯作者: Popa, Ionel