Different Photochemical Events of a Genetically Encoded Phenyl Azide Define and Modulate GFP Fluorescence
Different Photochemical Events of a Genetically Encoded Phenyl Azide Define and Modulate GFP Fluorescence
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DOI：
10.1002/anie.201301490
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Jones, D. Dafydd
中科院分区：
文献类型：
--
作者：
Reddington, Samuel C.;Rizkallah, Pierre J.;Jones, D. Dafydd
基因编码的蛋白质功能的光控制，称为光遗传学，[1]是一种以高时间和空间分辨率调节生物过程的强大方法。目前，光遗传学依赖于有限数量的辅因子依赖的光敏蛋白，如视蛋白通道[2]和LOV结构域[1b]，这可能会限制该方法的广泛应用。通过重新编程的遗传密码引入蛋白质序列固有的新的光化学[3]是一个有吸引力的替代方案。一种经典的非遗传方法包括使用苯叠氮化物作为光交联剂，在体外翻译后和非特异性引入。苯叠氮化物化学现在可以通过使用酪氨酸衍生物p-叠氮基-L-苯丙氨酸[4](图1a)在蛋白质中精确和确定的位置进行遗传编码，并已被用于定点修饰蛋白质。[5]然而，光化学效应在反应途径和用于控制蛋白质活性方面仍然相对未被探索。苯基叠氮化物的光解释放出氮气，形成反应性的单线态氮，它遵循几个特征路径(参见支持信息，图式S1)，其分布取决于局部环境、环取代基和温度。可能的途径包括还原为胺、C±H键插入或扩环。光控自体荧光蛋白目前是理想的高分辨率细胞成像的分子荧光剂。[6]人们致力于设计此类蛋白[6，7]，但仍然存在一些限制，包括碱性蛋白支架的稳定性和光转化特性。理想情况下，光控制将被设计成高度稳定的、单体的、快速折叠的自发荧光蛋白，如超级文件夹GFP(SfGFP)[8]，具有快速的光转化动力学和在相对较低的能量应用下的高幅度变化。在这里，我们展示了在定义的残基上加入单一的苯叠氮化合物化学单元在正常光稳定的sfGFP上注入光可控的荧光输出。三个残基作为AZF掺入的靶点(图1b)：Y66(SfGFPY66AzF)，构成部分
Genetically encoded photocontrol of protein function, known as optogenetics,[1] is a powerful approach for modulating biological processes with both high temporal and spatial resolution. Currently, optogenetics relies on a limited number of cofactor-dependent light sensitive proteins such as opsin channels [2] and LOV domains,[1b] which may restrict the general application of the approach. The introduction of new photochemistry intrinsic to the protein sequence through a reprogrammed genetic code [3] is an attractive alternative. A classic non-genetic approach involved the use of phenyl azides as photo-crosslinking agents introduced post-translationally and non-specifically in vitro. Phenyl azide chemistry can now be genetically encoded at precise and defined positions in a protein through the use of the tyrosine derivative p-azido-L-phenylalanine [4](Figure1a) and has been used to site-specifically modify proteins.[5] However, photochemical effects remain relatively unexplored both in terms of reaction pathways and use in controlling protein activity. Photolysis of phenyl azide releases N2 forming a reactive singlet nitrene that follows several characterized pathways (see Supporting Information, SchemeS1), the distribution of which depends on the local environment, ring-substituents, and temperature. Possible routes include reduction to an amine, CÀH bond insertion, or ring expansion.Photo-controllable autofluorescent proteins are currently desirable as molecular highlighters for high-resolution cell imaging.[6] Considerable effort is devoted to engineering such proteins [6, 7] but some limitations persist including robustness and photoconversion properties of the base protein scaffold. Ideally, photocontrol would be engineered into a highly stable, monomeric, fast-folding autofluorescent protein such as superfolder GFP (sfGFP)[8] with fast photoconversion kinetics and high magnitude changes on the application of relatively low energy. Here we show that incorporation of a single phenyl azide chemical unit at defined residues instils light-controllable fluorescence output over the normally photostable sfGFP. Three residues were targeted for AzF incorporation (Figure 1b): Y66 (sfGFPY66AzF) that forms part