Few methods allow determining the binding site of tightly binding ligands. We show that ligands containing a tert-butyl (e.g., Boc) group produce easily observable nuclear Overhauser effects (NOE) with the target protein even when the tert-butyl group is not highly solvent exposed. NOEs with methyl groups of the target protein are readily assigned by selectively isotope labeling, presenting a practical and quick way to pinpoint the location of the ligand without any prior specific nuclear magnetic resonance assignments of the protein. The approach works for nonexchanging ligands as well as for weakly binding ligands.

中文翻译：

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使用配体中的叔丁基来鉴定其在蛋白质上的结合位点

很少有方法可以确定紧密结合的配体的结合位点。我们表明，即使叔丁基没有高度暴露于溶剂中，含有叔丁基（例如Boc）的配体也会与目标蛋白产生易于观察到的核Overhauser效应（NOE）。可以通过选择性同位素标记轻松指定具有目标蛋白质甲基的NOE，从而提供了一种实用且快速的方法来精确定位配体的位置，而无需对该蛋白质进行任何先前的特定核磁共振分配。该方法适用于非交换配体以及弱结合配体。