Site-specific fluorescence double-labeling of proteins and analysis of structural changes in solution by Fluorescence Resonance Energy Transfer (FRET).
Site-specific fluorescence double-labeling of proteins and analysis of structural changes in solution by Fluorescence Resonance Energy Transfer (FRET).
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DOI：
10.1016/j.mex.2018.03.006
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发表时间：
2018
期刊：
影响因子：
1.9
通讯作者：
Biswas SB
中科院分区：
文献类型：
--
作者：
Patel MJ;Yilmaz G;Bhatia L;Biswas-Fiss EE;Biswas SB
插入 Cys4 序列和单个 Cys 基序允许使用蛋白质的 FlAsH 和 Alexa568 进行位点特异性标记。 FRET 是一项众所周知的技术，可用于分析用供体和受体荧光团标记的蛋白质的结构变化。荧光团间距离和峰强度的接近比用于了解结构变化。荧光共振能量转移 (FRET) 是一种众所周知的检测和定量溶液中蛋白质结构变化的方法。 FRET 需要用两个荧光团对位点特异性蛋白质进行标记，其中一个作为能量供体，另一个作为能量受体。然而，蛋白质的位点特异性标记通常复杂且困难，特别是在特定位点插入两个荧光团时。我们研究了几种蛋白质标记方法，并取得了不同程度的成功。这里描述的是一种双重标记策略，该策略在许多蛋白质靶标中可重复地发挥作用，我们相信该策略将适用于具有很少或没有天然半胱氨酸 (Cys) 残基的多种蛋白质。我们成功地双标记了炭疽杆菌的 DnaA 蛋白，该蛋白缺乏内在的 Cys 残基。通过体外诱变在 N 末端插入半胱氨酸残基，并通过 PCR 在 C 末端插入 Cys-Cys-Phe-Gly-Cys-Cys (CCPGCC) 序列。该蛋白在 CCPGCC 序列处用荧光素衍生物 FlAsH 进行位点特异性标记，随后在 N 末端 Cys 残基处用 Alexa568 马来酰亚胺标记。通过双标记蛋白质的 FRET 分析确定蛋白质与核苷酸、DNA 和抑制剂蛋白质结合的结构变化。这种用不同荧光团进行位点特异性蛋白质标记的综合新颖方法适用于理解不同的体外蛋白质组结构研究。在这里，我们以荧光团标记的 DnaA 蛋白构建体为例，描述了一种用于 FRET 光谱分析和结构变化定量评估的经过验证的技术。
Insertion of a Cys4 sequence and single Cys motif allowedsite-specific labeling with FlAsH and Alexa568 of a protein. FRET is a well-known technique useful for analysis of structural changes of proteins labeled with donor and accepter fluorophores. Inter-fluorophore distances and proximity ratios of the peakintensities are used to understand structural changes. Fluorescence Resonance Energy Transfer (FRET) is a well-known methodology for detection and quantitation of structural changes of proteins in solution. FRET requires site-specific protein labeling with two fluorophores, one of which functions as an energy donor and the other one as an energy acceptor. However, the site-specific labeling of protein is often complex and difficult, particularly when inserting two fluorophores in specific sites. We have examined several protein labeling approaches with a varying degree of success. Described here is a dual labeling strategy that worked reproducibly in a number of protein targets and we believe will be applicable to a variety of proteins, which have few or no native cysteine (Cys) residues. We have successfully double-labeled DnaA protein of Bacillus anthracis, which lacks intrinsic Cys residues. A cysteine residue was inserted at the N-terminus by in vitro mutagenesis and a Cys-Cys-Phe-Gly-Cys-Cys (CCPGCC) sequence at the C-terminus by PCR. This protein was labeled site-specifically with a fluorescein derivative, FlAsH, at the CCPGCC sequence followed by Alexa568 maleimide at the N-terminus Cys residue. Structural changes of the protein with nucleotide, DNA and an inhibitor protein binding were determined by FRET analysis of the double-labeled protein. This comprehensive novel methodology for site-specific protein labeling with different fluorophores is applicable for understanding different in vitro proteomic structural studies. Here, we describe a verified technique used for FRET spectral analysis and quantitative evaluation of structural changes using fluorophore labeled DnaA protein constructs as an example.