Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules.
Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules.
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DOI：
10.1002/cphc.201100897
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发表时间：
2012-03
期刊：
影响因子：
--
通讯作者：
Seidel CA
中科院分区：
文献类型：
--
作者：
Felekyan S;Kalinin S;Sanabria H;Valeri A;Seidel CA
介绍了一种寿命、偏振和光谱滤波的荧光相关光谱分析方法，简称滤波FCS（fFCS）。它使用，但不限于，多参数荧光检测，以区分分子种类之间的荧光寿命，偏振和光谱信息。像最近引入的荧光寿命相关光谱（FLCS）[Chem.Phys.Lett. 2002，353，439-445]，fFCS基于脉冲激光激发。然而，它使用物种特异性偏振和光谱分辨荧光衰减来产生滤波器。我们确定了最有效的方法来生成全局过滤器考虑到各向异性信息。因此，fFCS能够区分物种，即使它们具有非常接近或相同的荧光寿命，考虑到其他荧光参数的差异。fFCS可以作为一种工具，从不同物种的混合物中计算物种特异性自相关函数（SACF）和交叉相关函数（SCCF），以准确和定量地分析它们的浓度、扩散和动力学性质。计算的相关曲线也没有由非特异性背景信号引起的伪影。我们通过模拟仅由荧光各向异性的差异监测的配体-受体结合过程的极端情况来测试这种方法。此外，我们将fFCS应用于蛋白质Syntaxin-1的开放到闭合构象转变的实验性单分子FRET研究。总之，fFCS和SACF和SCCF的全局分析是在纳秒到毫秒的时间范围内研究生物分子的结合过程和构象动力学以及解开所涉及的分子状态的关键工具。
An analysis method of lifetime, polarization and spectrally filtered fluorescence correlation spectroscopy, referred to as filtered FCS (fFCS), is introduced. It uses, but is not limited to, multiparameter fluorescence detection to differentiate between molecular species with respect to their fluorescence lifetime, polarization and spectral information. Like the recently introduced fluorescence lifetime correlation spectroscopy (FLCS) [Chem. Phys. Lett. 2002, 353, 439–445], fFCS is based on pulsed laser excitation. However, it uses the species-specific polarization and spectrally resolved fluorescence decays to generate filters. We determined the most efficient method to generate global filters taking into account the anisotropy information. Thus, fFCS is able to distinguish species, even if they have very close or the same fluorescence lifetime, given differences in other fluorescence parameters. fFCS can be applied as a tool to compute species-specific auto- (SACF) and cross- correlation (SCCF) functions from a mixture of different species for accurate and quantitative analysis of their concentration, diffusion and kinetic properties. The computed correlation curves are also free from artifacts caused by unspecific background signal. We tested this methodology by simulating the extreme case of ligand–receptor binding processes monitored only by differences in fluorescence anisotropy. Furthermore, we apply fFCS to an experimental single-molecule FRET study of an open-to-closed conformational transition of the protein Syntaxin-1. In conclusion, fFCS and the global analysis of the SACFs and SCCF is a key tool to investigate binding processes and conformational dynamics of biomolecules in a nanosecond-to-millisecond time range as well as to unravel the involved molecular states.
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DOI：
10.1073/pnas.180317197
发表时间：
2000-09-12
影响因子：
11.1
作者：
Heinze, KG;Koltermann, A;Schwille, P
通讯作者：
Schwille, P
影响因子：
3.3
作者：
Kalinin, Stanislav;Valeri, Alessandro;Seidel, Claus A. M.
通讯作者：
Seidel, Claus A. M.
影响因子：
1.6
作者：
CHOWDHURY, FN;KOLBER, ZS;BARKLEY, MD
通讯作者：
BARKLEY, MD
影响因子：
2.4
作者：
Enderlein, J;Erdmann, R
通讯作者：
Erdmann, R
影响因子：
1.6
作者：
Enderlein, J;Gregor, I
通讯作者：
Gregor, I