Complex Fluorescence of the Cyan Fluorescent Protein: Comparisons with the H148D Variant and Consequences for Quantitative Cell Imaging
Complex Fluorescence of the Cyan Fluorescent Protein: Comparisons with the H148D Variant and Consequences for Quantitative Cell Imaging
复制标题
DOI：
10.1021/bi801400d
复制
发表时间：
2008-11-25
期刊：
影响因子：
2.9
通讯作者：
Pasquier, Helene
中科院分区：
文献类型：
--
作者：
Villoing, Aude;Ridhoir, Myriam;Pasquier, Helene
我们研究了纯化的增强型青色荧光蛋白（ECFP，具有发色团序列Thr-Trp-Gly）及其携带单一H148 D突变特征的更亮形式Cerulean的变体的荧光衰减。这两种蛋白质都表现出高度复杂的荧光衰减，显示出强烈的温度和pH依赖性。在中性pH下，H148 D突变导致（i）所有荧光寿命的普遍增加和（ii）亚群的消失，估计超过总ECFP分子的25%，其特征在于猝灭和红移荧光。ECFP及其H148 D突变体的荧光寿命分布在其他方面仍然非常相似，表明这两种蛋白质在其主要形式中具有高度的结构和动力学相似性。从热力学分析，我们得出结论，ECFP的多指数衰减不能简单地归因于，如通常所承认的，由NMR和X射线晶体学研究表征的缓慢构象交换[Seifert，M. H、等人（2002）J. Am. 124，7932-7942; Bae，J.H.，等人（2003）1 Mol. 328，1071-1081]。在活细胞中的平行测量表明，这些荧光性质在中性溶液中是非常相似的胞质ECFP。
We have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) 1 Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP.