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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基因编码的叠氮化苯的不同光化学事件定义和调节 GFP 荧光
DOI：
10.1002/ange.201301490
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发表时间：
2013
影响因子：
--
通讯作者：
Reddington S
中科院分区：
文献类型：
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作者：
Reddington S
蛋白质功能的遗传编码光控制，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性光敏蛋白，例如视蛋白通道[2]和LOV结构域[1b]，这可能会限制该方法的普遍应用。通过重新编程的遗传密码[3]将新的光化学引入蛋白质序列是一种有吸引力的选择。经典的非遗传方法涉及使用苯基叠氮化物作为光交联剂，在体外非特异性地在体外进行后处理。通过使用酪氨酸衍生物p-叠氮基-L-苯丙氨酸[4]，苯基叠氮化学现在可以在蛋白质中精确和确定的位置进行遗传编码（图1a），并已用于位点特异性修饰蛋白质。[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。苯基叠氮的光解释放N2，形成反应性单线态氮烯，其遵循几种特征路径（参见支持信息，方案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成胺、插入C2 H2OH键或扩环。光可控的自荧光蛋白是目前理想的高分辨率细胞成像的分子荧光剂。[6]相当大的努力致力于工程化这样的蛋白质[6，7]，但仍然存在一些限制，包括基础蛋白支架的鲁棒性和光转换特性。理想情况下，光控将被工程化为高度稳定的单体快速折叠的自体荧光蛋白，例如超折叠GFP（sfGFP）[8]，其具有快速的光转化动力学和在相对低的能量下的高幅度变化。在这里，我们表明，在定义的残基处并入单个苯基叠氮化学单元在通常光稳定的sfGFP上注入光可控的荧光输出。三个残基被靶向用于AzF掺入（图1b）：Y 66（sfGFPY 66 AzF），其形成部分
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