Bistable isoelectric point photoswitching in green fluorescent proteins observed by dynamic immunoprobed isoelectric focusing.
Bistable isoelectric point photoswitching in green fluorescent proteins observed by dynamic immunoprobed isoelectric focusing.
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DOI：
10.1021/ja3064292
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发表时间：
2012-10-24
影响因子：
15
通讯作者：
Herr AE
中科院分区：
文献类型：
--
作者：
Hughes AJ;Tentori AM;Herr AE
我们描述了一种新的等电点光开关现象，在野生型维多利亚水母（av）绿色荧光蛋白和E222 G突变体蓝水母（ac）绿色荧光蛋白。采用时间分辨微流控等电聚焦（IEF）和原位抗体印迹IEF的组合来监测暗（非荧光）和亮（荧光）GFP群体。通过IEF，观察到每个种群表现出不同的等电点（pI），因此表现出不同的形式静电荷。实验观察到的暗，较高的pI和明亮，较低的pI GFP种群之间的相互转换是严格控制的差分紫外线和蓝光曝光。推导了与可逆光漂白过程有关的电荷转移的化学计量和动力学。在音乐会与反应-传输模型的可逆电荷和荧光光开关，人口相互转换率的芯片上的测量表明，根据照明配置文件的静电电荷的GFPs的电阻和离散开关样调制的潜力。我们估计3-4个形式电荷区分avGFP的亮和暗群体，相比之下，acGFP的一个电荷。考虑到E222作为GFP β-桶内内部和出口氢键簇之间的桥梁的作用，两种突变体之间电荷转换幅度的差异为GFP结构内质子运输的质子线假说以及与本体溶剂的质子交换提供了有趣的证据。我们的轻便的动态和探测IEF测定应发现广泛用于光开关和其他电荷开关过程响应于刺激，包括光，温度，或结合/裂解事件的分析筛选和定量动力学分析。
We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the E222G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF were employed to monitor dark (non-fluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3–4 formal charges distinguish the bright and dark populations of avGFP, compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen bond clusters within the GFP β-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events.
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影响因子：
3.4
作者：
Agmon, N
通讯作者：
Agmon, N
影响因子：
15
作者：
Baranov, Mikhail S.;Lukyanov, Konstantin A.;Solntsev, Kyril M.
通讯作者：
Solntsev, Kyril M.
一系列独特的可逆可切换荧光蛋白，具有适合各种应用的有益特性
DOI：
10.1073/pnas.1113770109
发表时间：
2012-03-20
影响因子：
11.1
作者：
Chang, Hao;Zhang, Mingshu;Xu, Tao
通讯作者：
Xu, Tao
影响因子：
3.3
作者：
Agmon, Noam
通讯作者：
Agmon, Noam
影响因子：
64.8
作者：
Dickson, RM;Cubitt, AB;Moerner, WE
通讯作者：
Moerner, WE