Yu Yongsheng, Wang Jianpeng, Liu Jiahui, Ling Daishun, Xia Jiang
Department of Chemistry, Center of Novel Biomaterials, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, China.
PLoS One. 2015 Apr 15;10(4):e0122101. doi: 10.1371/journal.pone.0122101. eCollection 2015.
Natural proteins are often confined within their local microenvironments, such as three-dimensional confinement in organelles or two-dimensional confinement in lipid rafts on cytoplasmic membrane. Spatial confinement restricts proteins' entropic freedom, forces their lateral interaction, and induces new properties that the same proteins lack at the soluble state. So far, the phenomenon of environment-induced protein functional alteration still lacks a full illustration. We demonstrate here that engineered protein fragments, although being non-functional in solution, can be re-assembled within the nanometer space to give the full activity of the whole protein. Specific interaction between hexahistidine-tag (His-tag) and NiO surface immobilizes protein fragments on NiO nanoparticles to form a self-assembled protein "corona" on the particles inside the nanopores of mesoporous silica. Site-specific assembly forces a shoulder-by-shoulder orientation and promotes fragment-fragment interaction; this interaction together with spatial confinement of the mesopores results in functional re-assembly of the protein half fragments. To our surprise, a single half fragment of luciferase (non-catalytic in solution) exhibited luciferase activity when immobilized on NiO in the mesopores, in the absence of the complimentary half. This shows for the first time that spatial confinement can induce the folding of a half fragment, reconstitute the enzyme active site, and re-gain the catalytic capability of the whole protein. Our work thereby highlights the under-documented notion that aside from the chemical composition such as primary sequence, physical environment of a protein also determines its function.
天然蛋白质通常局限于其局部微环境中，例如在细胞器中的三维受限或在细胞质膜脂质筏中的二维受限。空间受限限制了蛋白质的熵自由度，迫使它们进行横向相互作用，并诱导出相同蛋白质在可溶状态下所缺乏的新特性。到目前为止，环境诱导蛋白质功能改变的现象仍缺乏完整的阐释。我们在此证明，工程化的蛋白质片段尽管在溶液中无功能，但可以在纳米空间内重新组装以赋予整个蛋白质完整的活性。六组氨酸标签（His标签）与NiO表面之间的特异性相互作用将蛋白质片段固定在NiO纳米颗粒上，从而在介孔二氧化硅纳米孔内的颗粒上形成自组装蛋白质“冠层”。位点特异性组装迫使片段肩并肩排列并促进片段间相互作用；这种相互作用与介孔的空间限制一起导致蛋白质半片段的功能重新组装。令我们惊讶的是，荧光素酶的单个半片段（在溶液中无催化活性）在不存在互补半片段的情况下固定在介孔中的NiO上时表现出荧光素酶活性。这首次表明空间限制可以诱导半片段折叠，重构酶活性位点，并恢复整个蛋白质的催化能力。我们的工作因此突出了一个记录不足的观点，即除了诸如一级序列等化学组成外，蛋白质的物理环境也决定其功能。
