Sakamoto Seiji, Terauchi Mika, Araki Yasuyuki, Wada Takehiko
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai, 980-8577, Japan.
Biopolymers. 2013 Nov;100(6):773-9. doi: 10.1002/bip.22304.
The design of proteins whose structure and function can be manipulated by the external stimuli has been of great interest in the field of protein engineering. In particular, caged proteins which can be activated by photo-irradiation become powerful tools for investigating a variety of biological events. Although protein caging is straightforward to render light-responsive protein functions, this approach mostly have difficulties based on the preparation of caged proteins in which amino acid residues required for biological activities must be specifically modified with synthetic photolabile groups. The synthetic peptide-based strategy for photoactivation of protein function may expand the versatility of protein caging approaches since the photolabile protecting group can be easily introduced into the peptide by means of standard solid-phase methods in a site-specific manner. In this study, we designed a new photoactivable green fluorescent protein (GFP), in which a relatively short C-terminal fragment (residues 214-230) of a dissected protein was modified with 7-diethylamino-4-hydroxymethylcoumarin (DECM) as a photoresponsive-protecting group. The introduced DECM unit completely inhibited the reconstitution with the GFP N-terminal fragment (residues 2-214). However, irradiation of visible light (>400 nm) resulted in efficient cleavage of DECM group, leading to acceleration of protein reassembly and concomitant GFP fluorescence recovery. These results demonstrated direct control of protein structure and function by application of the synthetic photocleavable functionality to a fragmented protein. The combined system of fragmented proteins and synthetic photocleavable elements will provide the useful and potentially wide applicable strategy for the regulation of protein structure and function by the light in a temporal and spacial manner.
其结构和功能可通过外部刺激进行操控的蛋白质设计，在蛋白质工程领域一直备受关注。特别地，可通过光照射激活的笼形蛋白成为研究各种生物事件的有力工具。尽管蛋白质笼化可直接赋予蛋白质光响应功能，但基于制备笼形蛋白的这种方法大多存在困难，因为生物活性所需的氨基酸残基必须用合成的光不稳定基团进行特异性修饰。基于合成肽的蛋白质功能光激活策略可能会扩展蛋白质笼化方法的通用性，因为光不稳定保护基团可通过标准固相方法以位点特异性方式轻松引入肽中。在本研究中，我们设计了一种新的光激活绿色荧光蛋白（GFP），其中将一种切割蛋白的相对较短的C端片段（残基214 - 230）用7 - 二乙氨基 - 4 - 羟甲基香豆素（DECM）作为光响应保护基团进行修饰。引入的DECM单元完全抑制了与GFP N端片段（残基2 - 214）的重组。然而，可见光（>400 nm）照射导致DECM基团的有效切割，从而加速蛋白质重组并伴随GFP荧光恢复。这些结果证明了通过将合成光可切割功能应用于片段化蛋白质来直接控制蛋白质的结构和功能。片段化蛋白质与合成光可切割元件的组合系统将为通过光以时间和空间方式调节蛋白质结构和功能提供有用且可能广泛适用的策略。
