Stabilization and Kinetics of an Adsorbed Protein Depends on the Poly(N-isopropylacrylamide) Grafting Density.
Stabilization and Kinetics of an Adsorbed Protein Depends on the Poly(N-isopropylacrylamide) Grafting Density.
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DOI：
10.1021/acs.biomac.1c00417
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发表时间：
2021-11-08
影响因子：
6.2
通讯作者：
Gruebele, Martin
中科院分区：
文献类型：
--
作者：
Mora-Sierra, Zully;Gopan, Gopika;Chang, Roger;Leckband, Deborah E.;Gruebele, Martin
聚（N-异丙基丙烯酰胺）（PNIPAM）在较低临界溶液温度（LCST，32°C）下的溶解度转变被广泛用作热开关，以快速、可逆地捕获和释放蛋白质和细胞。通常假设在 LCST 之上吸附到 PNIPAM 上的蛋白质不受聚合物相互作用的影响。在这里，我们表明，通过与 LCST 上方的末端接枝 PNIPAM 膜相互作用，磷酸甘油酸激酶的折叠稳定性显着增加。这种稳定性反映了蛋白质吸附对先前研究的接枝条件的依赖性。当蛋白质吸附到塌陷的聚合物“蘑菇”上时，就会实现最大程度的稳定。在较致密的聚合物“刷子”状态下，蛋白质稳定性下降至与聚合物自由表面无法区分的值，这与致密、塌陷的刷子的低吸附性一致。通过快速弛豫成像测量的动力学的完整温度依赖性表明 PNIPAM 不会影响折叠/展开机制。令人惊讶的是，对折叠/解折叠动力学的分析表明，PNIPAM 主要通过稳定蛋白质的折叠状态而不是破坏其解折叠状态来发挥作用。我们认为，聚合物的作用是通过与其表面相互作用来增加折叠蛋白质的构型熵，而不仅仅是通过拥挤未折叠状态。 FReI 样品池显示表面有 PNIPAM 聚合物以及与其相互作用的荧光标记蛋白质。
The solubility transition at the lower critical solution temperature (LCST, 32 °C) of poly(N-isopropyl acrylamide) (PNIPAM) is widely used as a thermal switch to rapidly, reversibly capture and release proteins and cells. It is generally assumed that proteins adsorbed to PNIPAM above the LCST are unaffected by polymer interactions. Here we show that the folding stability of the enzyme phosphoglycerate kinase is significantly increased by interactions with end-grafted PNIPAM films above the LCST. The stabilization mirrors the dependence of protein adsorption on grafting conditions studied previously. Maximum stabilization occurs when proteins adsorb to collapsed polymer ‘mushrooms’. In the denser polymer ‘brush’ regime, protein stabilization decreases back to a value indistinguishable from the polymer free surface, consistent with the low adsorption on dense, collapsed brushes. A full temperature dependence of the kinetics measured by Fast Relaxation Imaging reveals that PNIPAM does not affect the folding/unfolding mechanism. Surprisingly, analysis of the folding/unfolding kinetics suggests that PNIPAM acts mainly by stabilizing the folded state of the protein, not by destabilizing its unfolded state. We propose that the polymer acts by increasing the configurational entropy of the folded protein via interacting with its surface, rather than just by crowding the unfolded state. FReI sample cell showing PNIPAM polymer at the surface and fluorescent-labeled protein interacting with it.
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影响因子：
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