Martin Hugh, Kinns Helen, Mitchell Nick, Astier Yann, Madathil Rethi, Howorka Stefan
Department of Chemistry, University College London, London WC1H 0AJ, England, United Kingdom.
J Am Chem Soc. 2007 Aug 8;129(31):9640-9. doi: 10.1021/ja0689029. Epub 2007 Jul 18.
We describe nanoscale protein pores modified with a single hyperbranched dendrimer molecule inside the channel lumen. Sulfhydryl-reactive polyamido amine (PAMAM) dendrimers of generations 2, 3 and 5 were synthesized, chemically characterized, and reacted with engineered cysteine residues in the transmembrane pore alpha-hemolysin. Successful coupling was monitored using an electrophoretic mobility shift assay. The results indicate that G2 and G3 but not G5 dendrimers permeated through the 2.9 nm cis entrance to couple inside the pore. The defined molecular weight cutoff for the passage of hyperbranched PAMAM polymers is in contrast to the less restricted accessibility of flexible linear poly(ethylene glycol) polymers of comparable hydrodynamic volume. Their higher compactness makes sulfhydryl-reactive PAMAM dendrimers promising research reagents to probe the structure of porous membrane proteins with wide internal diameters. The conductance properties of PAMAM-modified proteins pores were characterized with single-channel current recordings. A G3 dendrimer molecule in the channel lumen reduced the ionic current by 45%, indicating that the hyperbranched and positively charged polymer blocked the passage of ions through the pore. In line with expectations, a smaller and less dense G2 dendrimer led to a less pronounced current reduction of 25%. Comparisons to recordings of PEG-modified pores revealed striking dissimilarities, suggesting that differences in the structural dynamics of flexible linear polymers vs compact dendrimers can be observed at the single-molecule level. Current recordings also revealed that dendrimers functioned as ion-selectivity filters and molecular sieves for the controlled passage of molecules. The alteration of pore properties with charged and hyperbranched dendrimers is a new approach and might be extended to inorganic nanopores with applications in sensing and separation technology.
我们描述了在通道腔内用单个超支化树枝状大分子修饰的纳米级蛋白质孔。合成了第2代、第3代和第5代巯基反应性聚酰胺胺（PAMAM）树枝状大分子，对其进行了化学表征，并使其与跨膜孔α-溶血素中的工程化半胱氨酸残基反应。使用电泳迁移率变动分析监测成功的偶联。结果表明，G2和G3树枝状大分子但不是G5树枝状大分子通过2.9nm的顺式入口渗透到孔内进行偶联。超支化PAMAM聚合物通过的定义分子量截止值与具有相当流体力学体积的柔性线性聚（乙二醇）聚合物的限制较少的可及性形成对比。它们更高的致密性使得巯基反应性PAMAM树枝状大分子成为探测具有宽内径的多孔膜蛋白结构的有前途的研究试剂。用单通道电流记录表征了PAMAM修饰的蛋白质孔的电导特性。通道腔内的一个G3树枝状大分子分子使离子电流降低了45%，表明超支化且带正电的聚合物阻止了离子通过孔。符合预期的是，较小且密度较低的G2树枝状大分子导致电流降低不太明显，为25%。与PEG修饰孔的记录进行比较揭示了显著差异，表明在单分子水平上可以观察到柔性线性聚合物与致密树枝状大分子在结构动力学上的差异。电流记录还表明，树枝状大分子起到了离子选择性过滤器和分子筛的作用，用于分子的受控通过。用带电和超支化树枝状大分子改变孔的性质是一种新方法，并且可能扩展到在传感和分离技术中有应用的无机纳米孔。
