FRET and Excitonic imaging of protein-protein interactions inside living cells

活细胞内蛋白质-蛋白质相互作用的 FRET 和激子成像

基本信息

批准号：
10915283
负责人：
Steven S Vogel
金额：
$ 231.9万
依托单位：
NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10915283
关键词：
Affinity Anisotropy Binding Biological Biology Biophotonics Biosensor Brain Ca(2+)-Calmodulin Dependent Protein Kinase Calcium Cells Circular Dichroism Collaborations Complex Coupling Cyclic AMP Dendritic Spines Energy Transfer Environment Evolution Fiber Fiber Optics Fluorescence Fluorescence Anisotropy Fluorescence Resonance Energy Transfer Glutamates Holoenzymes Homo Image Internet Laboratories Ligand Binding Ligands Measurement Measures Methodology Methods Microscopy Molecular Conformation Monitor Mus Mutagenesis National Institute of Child Health and Human Development Phosphotransferases Photometry Photons Physiological Protein Dynamics Proteins Quantum Mechanics Site Spectrum Analysis Speed Structure Synapses Temperature Theoretical Studies Time Universities biological systems calcium-dependent protein kinase calmodulin-dependent protein kinase II cost in vivo instrumentation interest multimodality optical fiber protein protein interaction quantum quantum computer red fluorescent protein time use

项目摘要

The objective of the Laboratory of Biophotonics and Quantum Biology is to develop new forms of microscopy and photometry to study protein-protein interactions under physiological conditions. Here we outline the current status of the three major projects in LBQB. LBQB has three Specific Aims: 1) Utilize Binary-FRET, our method for simultaneously monitoring two independent protein-protein interactions inside cells. We are using this approach to simultaneously monitor CaMKII activation and T-site ligand binding, to study CaMKII translocation into dendritic spines, as well as for studying conformational changes in EAAT-2 associated with glutamate transport. 2) To investigate the mechanism of ultrafast energy transfer between fluorescent proteins and understanding the basis for coherent excitonic interactions between fluorescent proteins. 3) To develop a methodology for monitoring protein-protein interactions using deep-brain fiber photometry. Specific Aim 1 This project is primarily under the direction of Dr. Nguyen, with collaborative support from Dr. Puhl. Dr. Nguyen has demonstrated that homo-FRET and hetero-FRET can be measured simultaneously and that these measurements can follow independent changes in the proximity of homo-FRET and hetero-FRET pairs. Dr. Nguyen has demonstrated the utility of this approach by simultaneously monitoring the binding of a CaM-Kinase-II T-site ligand using hetero-FRET while simultaneously measuring a conformational change in the kinase holoenzyme structure associated with ligand binding using homo-FRET. In addition to generating constructs to support Dr. Nguyen's project, Dr. Puhl is also currently developing binary biosensors that simultaneously monitor free calcium concentration with both low and high affinity to further demonstrate the utility of this approach. Specific Aim 2 Ms. Taumoefolau, in persuit of her thesis project, and in collaboration with Dr. Kim (University of Surrey) and Dr. Blank (NICHD) has been using time-resolved anisotropy, FCS, and antibunching to develop new analysis and instrumentation to investigate ultra-fast (faster than 140 ps) energy transfer between fluorescent proteins. We are also collaborating with Drs. Chen and Kliger at UC Santa Cruz to use CD spectroscopy to detect coherent interactions between paired fluorescent proteins, with Dr. Elisabetta Collini to use 2D ultra-fast spectroscopy to study red fluorescent protein dynamics, and with Dr. Alexandra Olaya-Castro to study the theoretical basis for strong coupling in fluorescent proteins. Our results indicate that a coherent energy transfer mechanism is responsible for this unexpected high-speed energy transfer. We speculate that such a mechanism might have utility in developing quantum computers. Specific Aim 3 In collaboration with Dr. Lovinger's laboratory (LIN) and Dr. Kim's Laboratory (university of Surrey) Drs. Nguyen is developing photon-efficient fiber optic-based instrumentation to monitor FRET-based biosensors for cAMP and A-kinase activity in living mice based on monitoring changes in fluorescence lifetime.

生物光谱和量子生物学实验室的目的是开发新形式的显微镜和光法以研究生理条件下的蛋白质 - 蛋白质相互作用。在这里，我们概述了LBQB三个主要项目的当前状态。 LBQB具有三个具体目标： 1）利用二进制 - fret，我们的方法同时监测细胞内部的两种独立的蛋白质蛋白质相互作用。我们正在使用这种方法同时监测CAMKII激活和T位配体结合，研究CaMKII转运为树突状棘，以及研究与谷氨酸转运相关的EAAT-2中的构象变化。 2）研究荧光蛋白之间超快能量转移的机制，并了解荧光蛋白之间相干激发相互作用的基础。 3）开发一种使用深脑纤维光度法监测蛋白质 - 蛋白质相互作用的方法。具体目的1该项目主要是在Nguyen博士的指导下，Puhl博士的合作支持。 Nguyen博士证明，可以同时测量Homo-Fret和Hetero-Fret，并且这些测量值可以遵循Homo-Fret和Hetero-Fret Pairs的接近性的独立变化。 Nguyen博士通过同时使用Hetero-fret监测Cam-激酶-II T位配体的结合，同时测量了使用HOMO-FRET与配体结合相关的激酶全酶结构的构象变化，从而证明了这种方法的实用性。除了生成构造以支持Nguyen博士的项目外，Puhl博士还正在开发二元生物传感器，这些二进制生物传感器同时监测具有低亲和力的自由钙浓度，以进一步证明这种方法的实用性。特定目的2 Taumoefolau女士，旨在人们物参与其论文项目，并与Kim博士（萨里大学）和Blank博士（NICHD）合作，一直使用时间分辨的各向异性，FCS和抗抗激素来开发新的分析和仪器，以研究超结合（比140 PS）能量转移荧光蛋白之间的超结合（FASTER）。我们还与Drs合作。 UC Santa Cruz的Chen和Kliger使用CD光谱法来检测配对的荧光蛋白之间的相互作用，与Elisabetta Collini博士使用2D超快速光谱学研究红色荧光蛋白动力学，并与AlexandraOlaya-Castro博士一起研究强大的COURORESCENTERISTIC。我们的结果表明，连贯的能量转移机制是导致这种意外的高速能量转移的原因。我们推测这种机制可能在开发量子计算机方面具有实用性。特定的目标3与Lovinger博士的实验室（Lin）和Kim博士的实验室（萨里大学）Drs合作。 Nguyen正在开发基于光纤的基于光纤的仪器，以根据荧光寿命的监测变化，监测活小鼠中的基于FRET的生物传感器和活小鼠中的A-激酶活性。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Measuring two-photon microscopy ultrafast laser pulse duration at the sample plane using time-correlated single-photon counting.

使用时间相关的单光子计数测量样品平面上的双光子显微镜超快激光脉冲持续时间。

DOI：
10.1117/1.jbo.25.1.014516
发表时间：
2020
期刊：
Journal of biomedical optics
影响因子：
3.5
作者：
Kim,Youngchan;Vogel,StevenS
通讯作者：
Vogel,StevenS

Anomalous surplus energy transfer observed with multiple FRET acceptors.

DOI：
10.1371/journal.pone.0008031
发表时间：
2009-11-25
期刊：
PloS one
影响因子：
3.7
作者：
Koushik SV;Blank PS;Vogel SS
通讯作者：
Vogel SS

Estimating the distance separating fluorescent protein FRET pairs.

DOI：
10.1016/j.ymeth.2013.06.021
发表时间：
2014-03-15
期刊：
METHODS
影响因子：
4.8
作者：
Vogel, Steven S.;van der Meer, B. Wieb;Blank, Paul S.
通讯作者：
Blank, Paul S.

Ultrafast fluorescence depolarisation in green fluorescence protein tandem dimers as hydrophobic environment sensitive probes.

DOI：
10.1039/d3cp01765f
发表时间：
2023-07-26
期刊：
Physical chemistry chemical physics : PCCP
影响因子：
0
作者：
通讯作者：

Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis.

使用荧光相关光谱和同源 FRET 分析破译 CaMKII 多聚化。

DOI：
10.1016/j.bpj.2017.02.005
发表时间：
2017
期刊：
Biophysical journal
影响因子：
3.4
作者：
Sarkar,Pabak;Davis,KaitlinA;Puhl3rd,HenryL;Veetil,JitheshV;Nguyen,TuanA;Vogel,StevenS
通讯作者：
Vogel,StevenS