Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates.
Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates.
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DOI：
10.1021/acs.biochem.1c00373
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发表时间：
2021-10-26
期刊：
影响因子：
2.9
通讯作者：
Hammer, Daniel A.
中科院分区：
文献类型：
--
作者：
Guan, Muyang;Garabedian, Mikael, V;Leutenegger, Marcel;Schuster, Benjamin S.;Good, Matthew C.;Hammer, Daniel A.
真核细胞将酶和其他细胞组分划分为不同的亚细胞区室，以产生专门的生化小生境。这些隔室的一个亚类在不存在脂质膜的情况下形成，通过蛋白质的液-液相分离形成生物分子缩合物或“无膜细胞器”，如核仁、应力颗粒和P体。由于它们能够从简单的起始材料形成区室，无膜细胞器是活细胞和原始细胞中工程化新功能的有吸引力的目标。在这项工作中，我们证明了新的酶活性与发光酶，NanoLuc，产生生物发光的蛋白质凝聚体的掺入。使用由C. elegans LAF-1，我们显示了体外具有酶活性的缩合物的功能化，并且定位于蛋白质凝聚体增强了分裂酶的组装和活性。为了构建起发光反应器作用的凝聚物，我们设计了一种侧翼有RGG结构域的NanoLuc酶。所得缩合物将NanoLuc浓缩为本体溶液的10倍，并显示出显著增加的净反应速率。我们进一步表明，凝聚粘度影响光发射由于扩散限制的行为。通过将NanoLuc酶拆分成其组成成分，我们证明NanoLuc活性可以通过共缩合重建。此外，我们证明了通过将NanoLuc靶向到体外凝聚物的表面来控制凝聚物内的酶的空间定位。总的来说，这项工作表明，无膜细胞器可以被赋予本地化的酶活性，这种活性可以通过酶重组和蛋白质表面活性剂的设计在空间和时间上进行控制。
Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or “membraneless organelles” such as nucleoli, stress granules, and P-bodies. Because of their ability to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of novel enzymatic activity in protein coacervates with a light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of C. elegans LAF-1, we show functionalization of condensates with enzymatic activity in vitro and that localization to protein coacervates enhances the assembly and activity of split enzymes. To build condensates that function as light emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. The resulting condensates concentrated NanoLuc by 10-fold over bulk solution and display significantly increased net reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. By splitting NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. Further, we demonstrate control of the spatial localization of enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity, and that this activity can be spatially and temporally controlled via enzyme reconstitution and design of protein surfactants.
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影响因子：
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