Self-Assembled Enzymatic Monolayer Directly Bound to a Gold Surface: Activity and Molecular Recognition Force Spectroscopy Studies
Self-Assembled Enzymatic Monolayer Directly Bound to a Gold Surface: Activity and Molecular Recognition Force Spectroscopy Studies
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DOI：
10.1021/ja205409v
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发表时间：
2011-08-31
影响因子：
15
通讯作者：
Tivanski, Alexei V.
中科院分区：
文献类型：
--
作者：
Ditzler, Lindsay R.;Sen, Arundhuti;Tivanski, Alexei V.
大肠杆菌二氢叶酸还原酶（ecDHFR）有一个表面半胱氨酸，C152，位于活性位点的对面和远端。在这里，我们表明，酶自发组装在超平的金表面作为一个均匀的，共价结合的单层。令人惊讶的是，发现通过射线照相分析测量的金固定的ecDHFR的活性与溶液中的游离酶的活性相似。用分子识别力谱研究了AFM探针固定的氨甲喋呤（MTX，一种紧结合的DHFR抑制剂）与金固定酶的解离力。用游离MTX处理ecDHFR单层减少了功能化尖端与表面的相互作用，表明这种相互作用确实是活性位点特异性的。这些研究结果表明，一个简单和直接的酶表面功能化的可行性，而不使用间隔，从而打开了大门，进一步应用在生物大分子力谱领域。
Escherichia coli dihydrofolate reductase (ecDHFR) has one surface cysteine, C152, located opposite and distal to the active site. Here, we show that the enzyme spontaneously assembles on an ultraflat gold surface as a homogeneous, covalently bound monolayer. Surprisingly, the activity of the gold-immobilized ecDHFR as measured by radiographic analysis was found to be similar to that of the free enzyme in solution. Molecular recognition force spectroscopy was used to study the dissociation forces involved in the rupture of AFM probe-tethered methotrexate (MTX, a tight-binding inhibitor of DHFR) from the gold-immobilized enzyme. Treatment of the ecDHFR monolayer with free MTX diminished the interaction of the functionalized tip with the surface, suggesting that the interaction was indeed active-site specific. These findings demonstrate the viability of a simple and direct enzymatic surface-functionalization without the use of spacers, thus, opening the door to further applications in the area of biomacromolecular force spectroscopy.