Biomacromolecules. 2021 Nov 8;22(11):4470-4478. doi: 10.1021/acs.biomac.1c00417. Epub 2021 Oct 4.
The solubility transition at the lower critical solution temperature (LCST, 32 °C) of poly(-isopropylacrylamide) (PNIPAM) is widely used as a thermal switch to rapidly and 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 (PGK) is increased by interactions with end-grafted PNIPAM films above the LCST. We systematically compare two protein mutants with different stabilities. The stabilization mirrors the degree of protein adsorption under grafting conditions studied previously. Maximum stabilization occurs when proteins adsorb to low density, collapsed polymer "mushrooms". In the denser polymer "brush" regime, protein stabilization decreases back to a value indistinguishable from the bulk solution, consistent with low protein adsorption on dense, collapsed brushes. The temperature-dependent kinetics measured by Fast Relaxation Imaging reveals that PNIPAM does not affect the overall folding/unfolding mechanism. Based on the different stabilizations of two mutants and the relaxation kinetics, we hypothesize that the polymer acts mainly by increasing the conformational entropy of the folded protein by interacting with the protein surface and less by crowding the unfolded state of PGK.
聚（异丙基丙烯酰胺）（PNIPAM）的低临界溶液温度（LCST，32°C）的溶解度转变被广泛用作热开关，以快速且可逆地捕获和释放蛋白质和细胞。通常假定在 LCST 以上吸附到 PNIPAM 上的蛋白质不受聚合物相互作用的影响。在这里，我们表明在 LCST 以上与端接枝 PNIPAM 薄膜的相互作用会增加酶磷酸甘油酸激酶（PGK）的折叠稳定性。我们系统地比较了两种稳定性不同的蛋白质突变体。在先前研究的接枝条件下，稳定化程度与蛋白质吸附程度相匹配。当蛋白质吸附到低密度、塌陷的聚合物“蕈”时，会出现最大稳定化。在更密集的聚合物“刷”区，蛋白质稳定化会回落到与体相溶液无法区分的水平，这与在密集、塌陷的刷上低蛋白质吸附一致。通过快速弛豫成像测量的温度依赖性动力学表明，PNIPAM 不会影响整体折叠/去折叠机制。基于两个突变体的不同稳定性和弛豫动力学，我们假设聚合物主要通过与蛋白质表面相互作用来增加折叠蛋白质的构象熵，而对 PGK 的去折叠状态的拥挤作用较小。
