荧光蛋白研究进展

生物物理学报2010年11月第26卷第11期:1025-1035

ACTA BIOPHYSICA SINICA 2010Vol.26No.11:1025-1035

荧光蛋白研究进展

杨杰，张智红，骆清铭

华中科技大学武汉光电国家实验室(筹)Britton Chance 生物医学光子学研究中心，武汉430074收稿日期：2010-08-15；接受日期：2010-10-28

基金项目：国家自然科学基金项目(30800208,30770525) ，新世纪优秀人才支持计划资助项目(NCET-08-0220)通讯作者：张智红，电话：(027)87792033，传真：(027)87792034，E-mail ：[email protected]

摘要：荧光蛋白在生物学众多研究领域中有着广泛的应用，基于荧光蛋白的分子探针和标记方法已成为活细胞或活体内动态成像研究生物大分子或细胞功能的重要工具。本文对现有荧光蛋白的种类和理化特性，及其在生物学研究中的应用进行了综述介绍。重点介绍了近年来荧光蛋白在亮度、Stokes 位移、光谱改变等方面的研究进展，介绍了光转换与光活化荧光蛋白及其在超分辨荧光成像技术中的应用。最后对荧光蛋白未来的发展方向进行了展望。

关键词：荧光蛋白；Stokes 位移；光转换与光活化；荧光显微成像；高分辨荧光成像中图分类号：Q-336

0引言

自1992年绿色荧光蛋白基因从水母体内克隆以来，现在已经从很多的海洋生物物种中克隆到了新的荧光蛋白，它们能特异地“点亮”生物分子或细胞，并显示出生物分子的活动情况，从而能更有助于我们揭示这些分子或细胞的活动规律及本质。已报道的荧光蛋白光谱分布于整个可见光区，它们被广泛应用于基因的表达调控、蛋白质空间定位与转运、蛋白折叠、信号传导、蛋白酶活性分析、生物分子相互作用等研究领域，荧光蛋白的发现与应用为现代生物学的研究提供了强有力的研究手段。Shimomura 首次从水母(Aequorea victoria ) 中分离出绿色荧光蛋白(green fluorescent protein ，GFP ) [1]，Chalfie 首次将GFP 的cDNA 转到新的物种中表达。Tsien 率先阐述了GFP 发光的化学机制[2]，并于1995年通过单点突变(S65T ) 技术获得了荧光强度和光稳定性大大增强的GFP 突变体(GFP-S65T )

[3]

。

Tsien 研究组基于GFP ，进一步突变出了蓝色荧光蛋白(blue fluorescent protein ，BFP ) 、青色荧光蛋白(cyan fluorescent protein ，CFP ) 和黄色荧光蛋白(yellow fluorescent protein ，YFP ) 。后来，研究人员又从珊瑚和海葵等物种中克隆出光谱红移的荧光蛋白，极大地扩展了荧光蛋白的多色成像应用[4,5]。近几年来，科学家巧妙地将光活化与光转换荧光蛋白应用于高分辨成像，突破光学衍射极限，分辨率达到几十纳米[6~10]，这是显微成像技术史上的一次革命性的飞跃。尽管这几年新的荧光蛋白层出不穷，但主要是围绕荧光蛋白的亮度、Stokes 位移(指激发峰与发射峰之间的距离) 和光谱特性的优化，以及新型光转换与光激活

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荧光蛋白分子的亮度由其量子产率与消光系数的乘积决定，与成像检测灵敏度密切相关，表1列出了一系列不同光谱特性的荧光蛋白。蓝色荧光蛋白EBFP (enhanced blue fluorescent protein ) 发光较弱，抗光漂白和抗酸性较差，用于细胞成像时背景信号高。几个课题组针对EBFP 开发了3个更亮的突变体：Azurite (亮度是EBFP 的1.6倍) [11]、EBFP2

[12]

，mTagBFP (亮度是EBFP 的3.7倍) [13]。最亮的蓝色荧光蛋白(亮度是EBFP 的2倍）

mTagBFP 是由红色荧光蛋白TagRFP 突变而来[13,14]，消光系数52000M -1cm -1，量子产率达到0.63，它的抗光漂白特性也有很大的提高。利用mTagBFP 细胞成像时大大提高了荧光的信号强度，并能与EGFP 组合形成优良的FRET (fluorescent resonant energy transfer ) 对[13]。

ECFP (enhanced cyan fluorescent protein ) 与EYFP (enhanced yellow fluorescent protein ) 组合是应用最为广泛的一对荧光共振能量转移FRET 对[15,16]。现在已经有很多新的

[17]

突变体在亮度上超越了ECFP ，如Cerulean 〔激发峰／发射峰：433/475(nm ) 〕、mTFP 〔激[18][19]发峰／发射峰：462/492(nm ) 〕、mTurquoise 〔激发峰／发射峰：433/475(nm ) 〕。mTFP

来源于Clavularia sp. 珊瑚的荧光蛋白突变体, 它的亮度是Cerulean 的两倍[20]。另外，通过建立寿命成像方法高通量地筛选青色荧光蛋白，有学者获得了几个量子产率提高的突变体，其中一个突变体命名为mTurquoise ，量子产率为0.84，亮度约是ECFP 的2倍[19]。

目前，绿光区荧光蛋白也有较多的突变体，但EGFP 仍然是最常用的，因为EGFP 除了高亮度外，还有很好的光稳定性。通过优化GFP 的折叠特性，有学者得到了一个具有超折叠能力的GFP Superfolder ，即使与不溶性蛋白质融合表达，Superfolder 仍然可以高效地折叠，从而保证其荧光亮度[20]。

由于早期获得的黄色荧光蛋白EYFP 已经很亮，现在仍被广泛使用。但EYFP 抗酸性差、对卤化物敏感，使它的应用受到限制[21]。在EYFP 基础上改进的突变体mCitrine [21]和mVenus [22]是目前应用最多的黄色荧光蛋白。然而，这两种黄色荧光蛋白与CFP 组合时FRET 动态范围仍然较小。因此，通过称为进化优化的方法对Caspase3生物传感器中配对—的CFP 和YFP 进行突变筛选，有研究组获得了动态范围最大的青/黄色FRET 对——CyPet/YPet[23]。但后来证实，YPet 与CyPet 在很大程度上是由于它们之间的二聚化作用导致了FRET 的增强[24,25]。其中，黄色荧光蛋白YPet 是目前报道最亮的。phiYFP 是一个更加红移的二聚化黄色荧光蛋白〔激发峰／发射峰：525/537(nm ) 〕，从Phialidium 水母中分离到，亮度与Venus 相当[26]。

对于橙色与红色系列荧光蛋白，较早报道的红色荧光蛋白(DsRed ) 的突变体有mBanana 、mOrange 、dTomato 、mTangerine 、mStrawberry 和mCherry (见表1) 。这些以水果命名的荧光蛋白表现出不同的光谱和理化特性，其中红色荧光蛋白mCherry 成熟快、单体特性好，已被广泛应用，但它的亮度较低[4]。从蘑菇珊瑚Fungia concinna 克隆的Kusabira Orange ，通过点突变，得到激发与发射峰分别为548和561nm 的单体荧光蛋白，命名为mKO [27]。对mKO 蛋白进一步优化，得到了成熟更快、亮度更高的突变体mKO κ[28]，这个新

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深蓝色蓝色系列

Sirius EBFP Azurite EBFP2TagBFP

青色系列

ECFP Cerulean CyPet mTurquoise mTFP1(Teal)Midoriishi-Cyan

绿色系列

UKG EGFP Emerald Superfolder

黄色系列

EYFP Venus Citrine YPet PhiYFP

橙色系列

mHoneydew mBanana mKO mKO κmOrange mOrange2

红色系列

TagRFP TagRFP 158T mRuby mCherry

深红色系列

Katushka mKate mKate2mPlum E2-Crimson mNeptune Tag657eqFP650eqFP670

近红外

mIFP

(nm)[***********][***********][***********]517525487/[***********][***********][***********]605684

(nm)[***********][***********][***********]530537537/[***********][***********][***********]670708

(l/mol·cm) [***********][***********][***********][***********][***********][***********][***********][***********][***********][***********][***********]00

0.240.30.550.560.630.40.60.510.840.850.90.720.60.680.650.60.60.80.770.40.10.70.60.610.70.600.480.410.350.20.670.330.40.10.230.20.10.240.060.07

(EGFP%)[***********][***********][**************]12.[***********][***********]4612.519

形式单体单体单体单体单体单体单体单体单体单体二聚体单体单体单体单体单体单体单体单体二聚体单体单体单体单体单体单体单体单体单体单体四聚体单体单体单体四聚体单体四聚体四聚体四聚体单体

[***********][***********][***********][***********]40

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而来的单体荧光蛋白mRuby 与TagRFP 具有高度的同源性，尽管在量子产率上比TagRFP 略低点，但是mRuby 具有更高的消光系数(112000M -1cm -1) ，这使它更适合做FRET 受体[33]。另外，mRuby 去掉了母体eqFP611的C 端SKL 序列(过氧化物酶体定位序列) ，可避免荧光蛋白定位到过氧化物酶体中。为了能将红色荧光蛋白用于长时间的成像观察，还筛选到了两种光稳定特别强的荧光蛋白mOrange2与TagRFP2，在光稳定性上比前体分别提高了25倍和9倍[32]。

2可见光光谱两端的荧光蛋白

由于荧光蛋白的发光团结构决定了它的光谱特性，从结构上来设计荧光蛋白的突变位点，有可能获得可见光光谱两端的荧光蛋白突变体(见表1) 。通过GFP-Phe66突变获得了一个深蓝色的荧光分子(激发峰为355nm 、发射峰为424nm ) ，但它的量子产率太低(0.013) 而无法使用[29]。进一步对GFP-Phe66突变T65Q 、Y145G 、H148S 、T203V 位点后，得到了深蓝色荧光蛋白Sirius ，亮度比GFP-Phe66提高了25倍[29]。

开发深红色或者近红外荧光蛋白对于活体生物成像更具价值。由于机体组织内的血红素在可见光波段有强吸收，而水和脂肪在红外波段有强吸收，仅在近红外光波段(650～900nm ) 的吸收系数最小且自发荧光最弱，被认为是活体成像的“光学窗口”[5,15]。早期，Tsien 等利用体细胞超突变方法对mRFP 进行突变，经过23轮的荧光分选，获得了激发峰向深红红移的mPlum

(激发峰为590nm 、发射峰为649nm ) ，但是mPlum 的亮度低，只有

EGFP 的10%[35]。mKate 是新型单体深红色荧光蛋白，由TagRFP 四个位点突变得到，它的激发峰波长在588nm ，发射峰波长在635nm ，亮度只有EGFP 的45%[5]，它的二聚化蛋白Katushka 亮度是EGFP 的67%[5]。与EGFP 和RFP 相比，mKate 和Katushka 用于活动物体内荧光成像时，成像深度更深，获得的光学信号更丰富，能清楚地观察转基因爪蟾表达Katushka 的肌肉组织[5]。mKate2是mKate V38A/S165A/K238R的突变体，亮度提高约一倍，激发峰/发射峰：但光谱没有发生改变[34]。对mKate2突变得到光谱红移的mNeptune 〔

[37]

600/650(nm ) 〕。对mKate2突变获得了更红移的TagRFP657，其激发与发射峰分别位于

611和657nm 处[38]，但是TagRFP657在亮度上远弱于mNeptune 。更红移的荧光蛋白能结合胆绿素发色团的近红外荧光蛋白突变单体IFP1.4，它的激发波长是684nm ，发射波长是708nm [40]。当他们利用腺病毒载体在小鼠肝脏表达这种突变体时，观察到强烈的近红外荧光，能更清楚地看到动物体内的肿瘤组织[40]。

3大Stokes 位移的荧光蛋白

表2列出大Stokes 位移的荧光蛋白。在多色成像时，大Stokes 位移可以有效地减少光谱的串扰。野生型的wtGFP 是一种Stokes 位移大的荧光蛋白，它的激发峰与发射峰分别在395和509nm [31]。但wtGFP 在475nm 仍有一个小的吸收峰，经过关键位点的突变(203Thr

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了一个新突变体mAmetrine ，它的激发与发射峰值分别为406和526nm [42]。mAmetrine 可与红色荧光蛋白tdTomato 组合形成FRET 对，并与另一对FRET 荧光蛋白mTFP/Citrine联合使用，可在同一细胞内进行双FRET 成像，实现两种分子事件的同步观察[42]。

表2大Stokes 位移的荧光蛋白及其特性

Table 2Properties of the fluorescent proteins with large stokes shift 名称激发峰发射峰消光系数量子产率聚合形式文献T-Sapphire [1**********]0.60单体41mAmetrine [1**********]0.58单体42mKeima [1**********]0.24单体43mLSS-Kate[1**********]00.08单体44mLSS-Kate2

460

605

26000

0.17

单体

mKeima 是最早报道的大Stokes 位移的红色荧光蛋白，它的发射与吸收峰分别位于440和620nm 处[43]。采用458nm 的光源同时激发CFP 与mKeima 两个荧光分子，能实现单光源的多色成像及荧光相关光谱(fluorescence correlation spectroscopy ，FCS ) 分析[43]。但mKeima 有两个激发峰，另一个在584nm 处，这不利于它的多色成像。为了克服这个缺点，最新报道的基于mKate 突变得到的LSS-mKate1(mKate/K69Y/P131T/S148G/M167E/T183S/M196V)

和LSS-mKate2(mKate/K69Y/P131T/S148G/S165A/M167D/

T183S/M196V) 两个突变体都只有一个吸收峰[44]。其激发峰与发射峰分别为463/624(nm) 和460/605(nm ) ，它们都具有好的抗酸性和单体特性，成熟快[44]。另外，LSS-mKate2亮度比mKeima 更高，利用它能实现双光子活体观察肿瘤细胞的转移与极性的变化[44]。

上面介绍的这些大Stokes 位移大的荧光蛋白，发光团都能发生激T203

发态质子转移(excited-state proton O transfer ，ESPT ) ，这主要是由于发N O N R

光团中的Tyr 与质子结合后产生H N

N R 的，如wtGFP 在分子内形成了氢键H148

H O H 的盐桥(图1) [45,46]。为了能从分子机O O O 理分析它们的这些规律，对wtGFP 146H H H

O H

O 采用飞秒受激拉曼光谱进行研究，E222

获取了具有飞秒时间分辨率的振动203

S205

光谱[47]，光谱显示了蛋白在构型变图1wtGFP 的激发态质子转移[46]E222是质子受体基团化过程中引起质子转移的过程，这

Fig.1Excited-state proton transfer in wild-type GFP [46]The proton-accepting group is E222

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4光转换与光活化荧光蛋白

4.1不可逆的光活化荧光蛋白

表3列出了光转换与光活化荧光蛋白，其中不可逆的光活化荧光蛋白分为两类：改变亮度的光活化荧光蛋白和改变颜色的光活化荧光蛋白。

表3光转换与光活化荧光蛋白及其特性

Table 3Properties of photoactivable and photoswitchable fluorescent proteins

分类不可逆的

名称

改变亮度的光活化荧光蛋白类PA-GFP(活化前) PA-GFP(活化后) PA-mCherry1(活化前) PA-mCherry1(活化后) 改变颜色的光活化荧光蛋白类Kaede(活化前) Kaede (活化后) PS-CFP2(活化前) PS-CFP2(活化后) mEosFP(活化前) mEosFP (活化后) Dendra2(活化前) Dendra2(活化后)

可逆的

Dronpa rsFastLime Padron bsDronpa Kindling

[***********][***********]580

[***********][***********]600

[***********][***********][***********][1**********]

0.880.330.20.230.640.620.50.550.850.770.640.50.07

四聚体四聚体单体单体单体单体单体单体单体单体单体单体四聚体

[***********]

[1**********]4

515517暗595

[***********]0

0.130.7900.46

单体单体单体单体

4945

激发峰

发射峰

消光系数

量子产率

聚合形式

文献

最早报道的光活化荧光蛋白，是对野生型wtGFP T203H 点突变后产生的PA-GFP ，属于不可逆的改变亮度的光活化荧光蛋白类[45]。它经过405nm 的紫色光激活后，再用488nm 的光激发成像，此时的发射光比激活前增强100倍，因而光活化区域与周边未活化区域形成了很高的荧光对比度，为活细胞内动态研究蛋白质分子的扩散运动提供了有效方法[36]。近年来发展的PALM (photoactivated localization microscopy ) 成像技术，使PA-GFP

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光照射来漂白这些已经定位的荧光分子，使它们不能被下一轮的激光再激活。这之后，分别用405和488nm 的激光来激活和漂白其他的荧光分子，进入下一次循环，这样的循环持续上百次后，可获得细胞内几乎所有荧光分子的精确定位，将这些分子的图像合成到一起就获得比传统光学显微镜分辨率高10倍以上的图像[45,49,58]。在PA-FP 蛋白被发现之后，又针对红色荧光蛋白突变获得了PA-mCherry1，应用PA-mCherry1与PA-GFP 实现了单细胞中双分子的超分辨成像[49]。

改变颜色的光活化荧光蛋白：1) PS-CFP2是可发生光谱迁移的光活化单体荧光蛋白，受405nm 激光激活后，其光发射由青色(468nm ) 转变为绿色(511nm ) ，激活后PS-CFP2的荧光强度在511nm 处增强了1500倍[51]。利用PS-CFP2标记的多巴胺转运受体，可检测多巴胺转运受体在细胞中的运动情况[51]。2) Kaede 是从石珊瑚(Lobophyllia hemprichii ) 中克隆的荧光蛋白，在日语中称Kaede 为“枫叶”。Kaede 在紫外光照射下其发射光能从绿色(518nm ) 变为红色(580nm ) [50]。Kaede 的光转换是由于紧邻发光团H62上的肽键发生断裂而引起的[50]。但是这个蛋白是一个四聚体，使其在蛋白标记与超分辨成像应用方面受到了限制。3) Eos 也是从石珊瑚克隆到的光活化蛋白，在390nm 的紫外光照射下由亮绿色荧光(516nm ) 变为橘红色荧光(581nm ) [59]，其机理同样也是光诱导导致与发光团相邻的肽链断裂而产生的光谱变化[59]。通过对EosFP 的突变得到了其单体荧光蛋白突变体，因而可用于超分辨显微成像研究蛋白质的空间定位及其功能[52]。4) Dendra-2是从Octocoral dendronephthy 中克隆到的、可被蓝光激活的单体荧光蛋白。其特性与Kaede 类似，激活后荧光不可逆地由绿色(发射峰为507nm ) 变成橙色(发射峰为573nm ) [53]，激活前后的对比度可达4500倍[53]。

4.2可逆的光转化荧光蛋白

最早获得的可逆光转化荧光蛋白是FP595，但是FP595的对比度很低，而且是四聚体[60]，它很快被其它蛋白(如Dronpa ) 所代替[54]。Dronpa 来源于Cnidaria ，它的激发与发射峰在503和518nm ，在强的蓝光(470～510nm ) 作用下使Dronpa 的荧光淬灭形成暗态的分子[54]。暗态的Dronpa 在390nm 处有吸收-O

Cis-Trans-峰，受到400nm 左右光照射时发生光活OH

化，可回到起初的绿色荧光态，这种转化可以反复多次进行[54]。Dronpa 的这种O

葑

O 明暗光转换机理是由于发光团的顺-反异N N

构化引起的，发光团处于顺式异构体时R R 2

R R 2

可发射荧光，当发光团Tyr 的羟基被质子On Off

化时，则处于反式异构体，表现为无荧光或暗态(图2) [61]。但利用Dronpa 成像图2Dropna 发光团顺反异构化[46]

时，其光转换速度偏慢[55]。研究人员开发

Fig.2cis -trans isomerism of a chromophore

of Dronpa [46]

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Padron 使用蓝光活化并成像，紫光淬灭。bsDronpa 是光谱蓝移的Dronpa 突变体，利用bsDronpa 与Dronpa 能实现双分子超分辨成像[56]。

Kindling 荧光蛋白(KFP1) 是从Anemonia sulcata 中克隆到的[57]。Kindling 荧光蛋白本身不能被激发出荧光，在低强度的绿光或黄光(525～580nm ) 照射下，荧光分子能转变成发红色荧光(600nm ) 。一旦停止照射，KFP1马上回复到暗态[57]。

5展望

本文主要介绍了荧光蛋白在亮度、Stokes 位移、光谱等特性方面的研究与发展，以及在光转换与光激活荧光蛋白及其在超分辨荧光成像技术中的应用。总体上讲，这些研究与应用已很深入，为生命科学研究提供了不可或缺的研究手段，但是很多方面仍然有待发展。目前活体成像深度仍有一定的局限性，为了提高深度成像的分辨率，需要优化出更合适于活体荧光成像的高亮度近红外荧光蛋白分子；另外，运用基于荧光蛋白的FRET 技术分析两种蛋白质之间的相互作用时，Foster 半径仍然太短，易导致FRET 信号太弱而无法检测到。此外，新型光学成像技术(如飞秒激光快速随机扫描双光子显微成像[62～64]、高分辨光声成像[65]、超分辨显微成像[58]等) 都迫切需要具备新特性的荧光分子与之相结合，以更大程度地提高成像的时空分辨率和检测灵敏度。我们有理由相信，荧光蛋白在今后的生物学研究中能发挥更为重要的作用。

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1034ACTA BIOPHYSICA SINICA ｜Vol.26No.11｜Nov. 2010

YANG Jie, ZHANG Zhihong, LUO Qingming

Britton Chance Biomedical Research Center, Huazhong University of Science and Technology, Wuhan 430074, China

This work was supported by grants from The National Natural Science Foundation of China(30800208,30770525）, Program for New Century Excellent Talents in University of China (NCET-08-0220)

Received:Agu 15, 2010Accepted:Oct 28, 2010

Corresponding author:ZHANG Zhihong, Tel:+86(27)87792033,Fax:+86(27)87792034,E-mail:[email protected]:Fluorescent proteins have been widely applied in biological research. The application of them became an important strategy for real-time imaging in living cells or in vivo . Here we gave a brief review about their types and physical chemistry properties and their application in biological research. We specially introduce some research progresses in respects to brightness, stocks shift and spectral properties. Photoactivable and photoswitchable fluorescent proteins and high-resolution fluorescent microscopy is also reviewed. At last, the fluorescent proteins were discussed with future perspectives.

Key Words:Fluorescent protein; Stokes shift; Photoactivable and photoswitchable; Fluorescent microscopy; High-resolution fluorescent imaging www.cjb.org.cn ｜ACTA BIOPHYSICA SINICA

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生物物理学报2010年11月第26卷第11期:1025-1035

ACTA BIOPHYSICA SINICA 2010Vol.26No.11:1025-1035

荧光蛋白研究进展

杨杰，张智红，骆清铭

华中科技大学武汉光电国家实验室(筹)Britton Chance 生物医学光子学研究中心，武汉430074收稿日期：2010-08-15；接受日期：2010-10-28

关键词：荧光蛋白；Stokes 位移；光转换与光活化；荧光显微成像；高分辨荧光成像中图分类号：Q-336

0引言

[3]

。

1025

[12]

，mTagBFP (亮度是EBFP 的3.7倍) [13]。最亮的蓝色荧光蛋白(亮度是EBFP 的2倍）

ECFP (enhanced cyan fluorescent protein ) 与EYFP (enhanced yellow fluorescent protein ) 组合是应用最为广泛的一对荧光共振能量转移FRET 对[15,16]。现在已经有很多新的

[17]

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深蓝色蓝色系列

Sirius EBFP Azurite EBFP2TagBFP

青色系列

ECFP Cerulean CyPet mTurquoise mTFP1(Teal)Midoriishi-Cyan

绿色系列

UKG EGFP Emerald Superfolder

黄色系列

EYFP Venus Citrine YPet PhiYFP

橙色系列

mHoneydew mBanana mKO mKO κmOrange mOrange2

红色系列

TagRFP TagRFP 158T mRuby mCherry

深红色系列

Katushka mKate mKate2mPlum E2-Crimson mNeptune Tag657eqFP650eqFP670

近红外

mIFP

(nm)[***********][***********][***********]517525487/[***********][***********][***********]605684

(nm)[***********][***********][***********]530537537/[***********][***********][***********]670708

(l/mol·cm) [***********][***********][***********][***********][***********][***********][***********][***********][***********][***********][***********]00

0.240.30.550.560.630.40.60.510.840.850.90.720.60.680.650.60.60.80.770.40.10.70.60.610.70.600.480.410.350.20.670.330.40.10.230.20.10.240.060.07

(EGFP%)[***********][***********][**************]12.[***********][***********]4612.519

[***********][***********][***********][***********]40

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2可见光光谱两端的荧光蛋白

(激发峰为590nm 、发射峰为649nm ) ，但是mPlum 的亮度低，只有

[37]

600/650(nm ) 〕。对mKate2突变获得了更红移的TagRFP657，其激发与发射峰分别位于

3大Stokes 位移的荧光蛋白

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表2大Stokes 位移的荧光蛋白及其特性

460

605

26000

0.17

单体

和LSS-mKate2(mKate/K69Y/P131T/S148G/S165A/M167D/

上面介绍的这些大Stokes 位移大的荧光蛋白，发光团都能发生激T203

发态质子转移(excited-state proton O transfer ，ESPT ) ，这主要是由于发N O N R

光团中的Tyr 与质子结合后产生H N

N R 的，如wtGFP 在分子内形成了氢键H148

H O H 的盐桥(图1) [45,46]。为了能从分子机O O O 理分析它们的这些规律，对wtGFP 146H H H

O H

O 采用飞秒受激拉曼光谱进行研究，E222

获取了具有飞秒时间分辨率的振动203

S205

光谱[47]，光谱显示了蛋白在构型变图1wtGFP 的激发态质子转移[46]E222是质子受体基团化过程中引起质子转移的过程，这

Fig.1Excited-state proton transfer in wild-type GFP [46]The proton-accepting group is E222

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4光转换与光活化荧光蛋白

4.1不可逆的光活化荧光蛋白

表3列出了光转换与光活化荧光蛋白，其中不可逆的光活化荧光蛋白分为两类：改变亮度的光活化荧光蛋白和改变颜色的光活化荧光蛋白。

表3光转换与光活化荧光蛋白及其特性

Table 3Properties of photoactivable and photoswitchable fluorescent proteins

分类不可逆的

名称

可逆的

Dronpa rsFastLime Padron bsDronpa Kindling

[***********][***********]580

[***********][***********]600

[***********][***********][***********][1**********]

0.880.330.20.230.640.620.50.550.850.770.640.50.07

四聚体四聚体单体单体单体单体单体单体单体单体单体单体四聚体

[***********]

[1**********]4

515517暗595

[***********]0

0.130.7900.46

单体单体单体单体

4945

激发峰

发射峰

消光系数

量子产率

聚合形式

文献

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ACTA BIOPHYSICA SINICA ｜Vol.26No.11｜Nov. 2010

4.2可逆的光转化荧光蛋白

Cis-Trans-峰，受到400nm 左右光照射时发生光活OH

化，可回到起初的绿色荧光态，这种转化可以反复多次进行[54]。Dronpa 的这种O

葑

O 明暗光转换机理是由于发光团的顺-反异N N

构化引起的，发光团处于顺式异构体时R R 2

R R 2

可发射荧光，当发光团Tyr 的羟基被质子On Off

化时，则处于反式异构体，表现为无荧光或暗态(图2) [61]。但利用Dronpa 成像图2Dropna 发光团顺反异构化[46]

时，其光转换速度偏慢[55]。研究人员开发

Fig.2cis -trans isomerism of a chromophore

of Dronpa [46]

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Padron 使用蓝光活化并成像，紫光淬灭。bsDronpa 是光谱蓝移的Dronpa 突变体，利用bsDronpa 与Dronpa 能实现双分子超分辨成像[56]。

5展望

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1034ACTA BIOPHYSICA SINICA ｜Vol.26No.11｜Nov. 2010

YANG Jie, ZHANG Zhihong, LUO Qingming

Britton Chance Biomedical Research Center, Huazhong University of Science and Technology, Wuhan 430074, China

This work was supported by grants from The National Natural Science Foundation of China(30800208,30770525）, Program for New Century Excellent Talents in University of China (NCET-08-0220)

Received:Agu 15, 2010Accepted:Oct 28, 2010

Key Words:Fluorescent protein; Stokes shift; Photoactivable and photoswitchable; Fluorescent microscopy; High-resolution fluorescent imaging www.cjb.org.cn ｜ACTA BIOPHYSICA SINICA

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