Arginine- and lysine-specific polymers for protein recognition and immobilization
Arginine- and lysine-specific polymers for protein recognition and immobilization
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DOI：
10.1021/ja0560229
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发表时间：
2006-01-18
影响因子：
15
通讯作者：
Schrader, T
中科院分区：
文献类型：
--
作者：
Renner, C;Piehler, J;Schrader, T
基于甲基丙烯酰胺的二膦酸盐的自由基聚合将弱精氨酸结合物转变为强大的聚合蛋白受体。通过简单的共聚方案即可获得具有优异水溶性的丹磺酰标记的均聚物和共聚物。模型研究指出，刚性棒状致密堆积的均聚物 1 和具有空间分离的双膦酸酯单元的柔性共聚物 2 之间存在显着的结构差异。缓冲水溶液 (pH = 7.0) 中的荧光滴定证实均聚物对寡精氨酸肽具有优异的亲和力，Tat 肽达到纳摩尔 K-D 值。碱性蛋白质与 1 和 2 的结合几乎同样良好，具有微摩尔亲和力，后者产生更多可溶性复合物。单体的 Arg 选择性转移到聚合物上，与具有相当 pl、大小和（Arg/Lys 与 Glu/Asp）比率的富含赖氨酸悬垂物相比，该聚合物与富含 Arg 的蛋白质结合紧密 1 个数量级。两种聚合物通过聚乙烯亚胺层非共价沉积在玻璃基板上，产生适合肽和蛋白质固定的新材料。 RlfS 测量可以计算缔合常数 K-a 以及解离动力学 k(D)。他们通常证实了免费解决方案中已经发现的趋势。对静电势表面的仔细检查表明，碱性结构域有利于蛋白质在平坦表面上的结合。通过与聚硫酸乙烯酯的比较，证明了双膦酸盐聚合物对碱性蛋白质的高特异性，而聚硫酸乙烯酯在 RlfS 实验中几乎没有影响。因此，几种不同的共聚单体单元的共聚而不发生交联能够实现游离溶液中碱性蛋白质的表面识别以及它们在表面上的有效固定。
Free radical polymerization of methacrylamide-based bisphosphonates turns weak arginine binders into powerful polymeric protein receptors. Dansyl-labeled homo- and copolymers with excellent water solubility are accessible through a simple copolymerization protocol. Modeling studies point to a striking structural difference between the stiff rodlike densely packed homopolymer 1 and the flexible copolymer 2 with spatially separated bisphosphonate units. Fluorescence titrations in buffered aqueous solution (pH = 7.0) confirm the superior affinity of the homopolymer toward oligoarginine peptides reaching nanomolar K-D values for the Tat peptide. Basic proteins are bound almost equally well by 1 and 2 with micromolar affinities, with the latter producing much more soluble complexes. The Arg selectivity of the monomer is transferred to the polymer, which binds Arg-rich proteins 1 order of magnitude tighter than lysine-rich pendants of comparable pl, size, and (Arg/Lys vs Glu/Asp) ratio. Noncovalent deposition of both polymers on glass substrates via polyethyleneimine layers results in new materials suitable for peptide and protein immobilization. RlfS measurements allow calculation of association constants K-a as well as dissociation kinetics k(D). They generally confirm the trends already found in free solution. Close inspection of electrostatic potential surfaces suggest that basic domains favor protein binding on the flat surface. The high specificity of the bisphosphonate polymers toward basic proteins is demonstrated by comparison with polyvinyl sulfate, which has almost no effect in RlfS experiments. Thus, copolymerization of few different comonomer units without cross-linking enables surface recognition of basic proteins in free solution as well as their effective immobilization on surfaces.