A noncovalent switch for Lysozyme
A noncovalent switch for Lysozyme
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DOI：
10.1021/ja075507p
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发表时间：
2007-12-26
影响因子：
15
通讯作者：
Schrader, Thomas
中科院分区：
文献类型：
--
作者：
Wenck, Kirstin;Koch, Sebastian;Schrader, Thomas
提出了一种外部控制蛋白质活性的新概念，并以人工溶菌酶开关为例进行了论证。针对活性位点周围氨基酸残基的甲基丙烯酰胺共聚单体单元的自由基共聚，提供了能够以高效的剂量依赖方式抑制酶活性的聚合蛋白宿主（IC50约为1.0，相当于约为0.7 μ M）。聚合物上的所有结合位点类型通过静电吸引、疏水力和底物模拟协同工作。在原生凝胶电泳中，定义明确的2:1复合物（聚合物/蛋白质）迁移到阳极。即使在250 mM NaCl下，10倍的聚合物过量也能完全停止细菌细胞壁的降解。动力学研究指出了竞争机制（Lineweaver - Burk图）。纯溶菌酶及其聚合物配合物的CD光谱难以区分；再加上完全缺乏达到最大抑制作用的预孵育时间，因此实验证据表明三级酶结构得到了保存——没有发生变性。在酶-聚合物络合物中加入优质的络合剂聚精氨酸，使抑制剂从蛋白质表面轻度分离，使酶活性恢复90%。因此，溶菌酶可以通过连续添加聚合物抑制剂、聚精氨酸和聚合物再次被关闭、打开和关闭。
A new concept for the external control of protein activity is presented and demonstrated on the example of an artificial Lysozyme switch. Radical copolymerization of selected methacrylamide-based comonomer units tailored for amino acid residues surrounding the active site furnishes polymeric protein hosts that are able to inhibit enzymatic activity in a highly efficient dose-dependent manner (IC50 approximate to 1.0 equiv approximate to 0.7 mu M). All binding site types on the polymer work cooperatively, using electrostatic attraction, hydrophobic forces, and substrate mimicry. In a native gel electrophoresis, the well-defined 2:1 complex (polymer/protein) migrates to the anode. Even at 250 mM NaCl, a 10-fold polymer excess is able to shut down bacterial cell wall degradation completely. A kinetic investigation points to a competitive mechanism (Lineweaver - Burk plots). CD spectra of pure Lysozyme and its polymer complex are indistinguishable; together with a total lack of preincubation time for maximum inhibition, experimental evidence is thus produced for a preserved tertiary enzyme structure-no denaturation occurs. Addition of the superior complexing agent polyarginine to the enzyme-polymer complex mildly detaches the inhibitor from the protein surface and leads to 90% recovery of enzymatic activity. Thus, Lysozyme could be turned off, on, and off again by consecutive addition of the polymeric inhibitor, polyarginine, and polymer again.