Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle.
Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle.
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DOI：
10.1039/c6sc00944a
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发表时间：
2016-10-01
期刊：
影响因子：
8.4
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Hartley AM;Worthy HL;Reddington SC;Rizkallah PJ;Jones DD
通过将单个苯基叠氮基团在残基148处遗传并入超折叠GFP（sfGFP）中，我们提供了这种高度通用的化学手柄如何通过光化学或生物缀合在体外和体内用于积极切换蛋白质功能的分子描述。通过将单个苯基叠氮基团在残基148处遗传并入超折叠GFP（sfGFP）中，我们提供了这种高度通用的化学手柄如何通过光化学或生物缀合在体外和体内用于积极切换蛋白质功能的分子描述。用对叠氮基-L-苯丙氨酸（azF）取代H148通过破坏定义发色团荷电状态的H-键和质子转移网络使主激发峰蓝移约90 nm。用简单的二苄基环辛炔或UV照射的生物正交点击修饰改变中性-阴离子发色团平衡，将荧光切换到最佳的λ 490 nm激发。点击修饰也提高了量子产率超过未修饰的和原始的蛋白质。点击改性和光化学转化形式的晶体结构表明，功能切换是由于局部构象的变化，优化周围的发色团的相互作用网络。经辐照的蛋白质的晶体结构和质谱研究表明，苯基叠氮化物转化为脱氢氮杂卓和/或氮杂卓酮。因此，蛋白质包埋的苯基叠氮化物可以用于超越简单的光交联剂和被动共轭手柄，并模仿许多自然的翻译后修饰：调制虽然在相互作用网络的变化。
Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function in vitro and in vivo via either photochemistry or bioconjugation. Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function in vitro and in vivo via either photochemistry or bioconjugation. Replacement of H148 with p-azido-l-phenylalanine (azF) blue shifts the major excitation peak ∼90 nm by disrupting the H-bond and proton transfer network that defines the chromophore charged state. Bioorthogonal click modification with a simple dibenzylcyclooctyne or UV irradiation shifts the neutral-anionic chromophore equilibrium, switching fluorescence to the optimal ∼490 nm excitation. Click modification also improved quantum yield over both the unmodified and original protein. Crystal structures of both the click modified and photochemically converted forms show that functional switching is due to local conformational changes that optimise the interaction networks surrounding the chromophore. Crystal structure and mass spectrometry studies of the irradiated protein suggest that the phenyl azide converts to a dehydroazepine and/or an azepinone. Thus, protein embedded phenyl azides can be used beyond simple photocrosslinkers and passive conjugation handles, and mimic many natural post-translational modifications: modulation though changes in interaction networks.