蛋白质-配体相互作用是分子识别的亮点之一。这种类型的相互作用的最普遍的应用是药物开发,其需要与靶蛋白结合的配体的高通量筛选。我们的目标是通过简单的检测找到一种结合配体,一旦发现了这种配体,然后可以使用其他方法来测量详细的动力学或热力学参数。我们从这样的想法开始,即如果配体NMR信号结合到非翻滚团块,则它将消失。为了创造不翻滚的质量,我们尝试了目标蛋白的聚集体,与弹性蛋白样多肽融合。我们选择麦芽糖结合蛋白作为测试案例,我们尝试了几种糖,其中包括麦芽糖,葡萄糖,蔗糖,乳糖,半乳糖,麦芽三糖,和β-环糊精。H-1NMR光谱中的麦芽糖信号完全消失,如所希望的,在蛋白质聚集的298K时,蛋白质与配体的比例为1:3。除了快速移动的部分,蛋白质信号在聚集时也消失了,这导致比单体形式更干净的背景。因为我们只需要在混合物中寻找消失的信号,它在高通量筛选中应该是有用的。除麦芽三糖和β-环糊精外,其他类型的糖,是麦芽糖的兄弟姐妹,似乎根本没有绑定。我们相信我们的系统在处理较小的目标蛋白时会特别有效,所以只有当聚集体形成时,蛋白质和结合的配体才会失去信号。我们希望我们提出的方法将有助于通过同时直接从混合物中鉴定几种粘合剂来加速有效候选药物的开发。
Protein-ligand interaction is one of the highlights of molecular recognition. The most popular application of this type of interaction is drug development which requires a high throughput screening of a ligand that binds to the target protein. Our goal was to find a binding ligand with a simple detection, and once this type of ligand was found, other methods could then be used to measure the detailed kinetic or thermodynamic parameters. We started with the idea that the ligand NMR signal would disappear if it was bound to the non-tumbling mass. In order to create the non-tumbling mass, we tried the aggregates of a target protein, which was fused to the elastin-like polypeptide. We chose the maltose binding proteinas a test
case, and we tried it with several sugars, which included maltose, glucose, sucrose, lactose, galactose, maltotriose, and β-cyclodextrin. The maltose signal in the H-1 NMR spectrum disappeared completely as hoped around the protein to ligand ratio of 1:3 at 298 K where the proteins aggregated. The protein signals also disappeared upon aggregation except for the fast-moving part, which resulted in a cleaner background than the monomeric form. Since we only needed to look for a disappearing signal amongst those from the mixture, it should be useful in high throughput screening. Other types of sugars except for the maltotriose and β-cyclodextrin, which are siblings of the maltose, did not seem to bind at all. We believe that our system would be especially more effective when dealing with a smaller target protein, so both the protein and the bound ligand would lose their signals only when the aggregates formed. We hope that our proposed method would contribute to accelerating the development of the potent drug candidates by simultaneously identifying several binders directly from a mixture.