Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy
Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy
复制标题
DOI：
10.1016/s0006-3495(97)78307-3
复制
发表时间：
1997-11-01
影响因子：
3.4
通讯作者：
Piston, DW
中科院分区：
文献类型：
--
作者：
Patterson, GH;Knobel, SM;Piston, DW
我们已经调查了相关的定量成像在活细胞中的五个绿色荧光蛋白（GFP）的变体，已被广泛使用或潜在的有用的属性。我们测量了wtGFP、alpha GFP（F99 S/M153 T/V163 A）、S65 T、EGFP（F64 L/S65 T）和蓝移变体EBFP（F64 L/S65 T/Y 66 H/Y145 F）的消光系数、量子产率、pH效应、光漂白效应和温度依赖性发色团形成。吸光度和荧光光谱显示wtGFP和α GFP的消光系数和量子产率之间几乎没有差异。相反，S65 T和EGFP消光系数使它们在488 nm激发时都比wtGFP亮6倍，并且EBFP在近UV波长区域激发时比wtGFP吸收更强，尽管它具有低得多的量子效率。当在488 nm激发时，GFP都比荧光素更耐光漂白。然而，wtGFP和α GFP光漂白模式显示由蛋白质发色团的光转化引起的荧光发射的初始增加。当在395 nm激发时，wtGFP荧光比488 nm更快地降低，但当在该波长激发时，它仍然比EBFP更耐光。wtGFP和alpha GFP在较宽的pH范围内非常稳定，但其他变体的荧光在pH 7以下迅速下降。当在细菌中表达时，发现wtGFP和S65 T中的发色团形成效率在37摄氏度下低于28摄氏度，但其他三种变体在37摄氏度和28摄氏度之间几乎没有差异。总之，没有单一的GFP变体是每种应用的理想选择，但每一种变体在活细胞定量成像方面都有优缺点。
We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alpha GFP (F99S/ M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alpha GFP, In contrast, S65T and EGFP extinction coefficients made them both similar to 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein, However, the wtGFP and alpha GFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alpha GFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7, When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.