Borst J W, Laptenok S P, Westphal A H, Kühnemuth R, Hornen H, Visser N V, Kalinin S, Aker J, van Hoek A, Seidel C A M, Visser A J W G
Microspectroscopy Centre, Laboratories of Biochemistry and Biophysics, Wageningen University, Wageningen, The Netherlands.
Biophys J. 2008 Dec;95(11):5399-411. doi: 10.1529/biophysj.107.114587. Epub 2008 Sep 12.
Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.
荧光共振能量转移（FRET）是一种广泛应用的方法，用于监测与合适的供体 - 受体对共轭的生物大分子之间或内部的相互作用。通常通过测量在不存在和存在受体分子的情况下供体荧光寿命来观察FRET。然而，这些寿命可能源自相互作用和非相互作用的分子，这妨碍了对FRET数据的定量解释。我们描述了一种检测FRET的方法，该方法通过监测供体激发时受体荧光的上升时间来检测仅经历FRET的那些分子。与更常用的供体荧光猝灭方法相比，该方法的一大优势在于，即使在FRET效率接近100％导致供体荧光高度猝灭的情况下，也能准确测定FRET的转移速率。随后，通过在供体激发和受体检测的相同条件下进行的时间相关荧光各向异性测量，获得供体和受体发色团之间的相对取向。使用基于FRET的钙传感器黄变色龙3.60（YC3.60），因为它在结合钙时会改变其构象，从而提高FRET效率。在绘制YC3.60中FRET部分之间的距离和取向角之后，可以创建有钙和无钙情况下该FRET传感器的卡通模型。对这些表示的独立支持来自于在选择性激发受体的情况下，在整体和单分子条件下测定YC3.60的流体动力学性质的实验。从荧光相关光谱法以及通过荧光各向异性衰减分析一致发现的旋转扩散时间可以得出结论，开放结构（无钙）是灵活的，而封闭构象则相当刚性。两种独立方法的结合给出了一致的结果，并提供了一种快速且特定的方法来分析蛋白质在配体结合时的结构和动态变化。
