Laboratory of Biophysics, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands.
J Phys Chem B. 2012 Mar 8;116(9):3013-20. doi: 10.1021/jp211830e. Epub 2012 Feb 22.
Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time- and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca(2+) concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.
YC3.60（Yellow Cameleon 3.60）是一种基于Förster 共振能量转移（FRET）的钙传感器。该传感器由钙调蛋白结构域和 M13 肽组成，位于增强型青色荧光蛋白（ECFP）和增强型黄色荧光蛋白（EYFP）的变体 Venus 之间。根据钙离子浓度的不同，从供体 ECFP 到受体 EYFP 的 FRET 效率会发生变化。在这项研究中，我们使用皮秒时间分辨率，在水溶液中记录了 ECFP、EYFP 和 YC3.60 的时间分辨荧光光谱，使用了不同的激发波长。通过对纯化的 YC3.60 在无钙和结合钙两种构象下的时间和波长分辨荧光的全局和目标分析，深入了解了 FRET 动力学。结果清楚地表明，对于这两种构象，都存在两种不同的供体群体：一种主要的供体能够发生 FRET，而另一种次要的供体则不能发生 FRET。结合钙构象的转移时间为 21 ps，而无钙构象的转移时间则在 1 ns 左右。YC3.60 的受体和供体荧光强度的比率成像通常用于测量活细胞中的 Ca(2+)浓度。从获得的结果可以清楚地看出，强度比受到不参与 FRET 的供体分子的存在的强烈影响，从而显著影响结果的定量解释。
