Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance
Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance
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DOI：
10.1021/ac0105888
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发表时间：
2001-12-01
影响因子：
7.4
通讯作者：
Pitner, JB
中科院分区：
文献类型：
--
作者：
Gestwicki, JE;Hsieh, HV;Pitner, JB
小分子很难用市售的表面等离子体共振（SPR）仪器直接检测。这是因为低分子量化合物没有足够的质量来引起折射率的可测量变化。然而，除了分析物的质量外，折射率对其他性质也很敏感。近年来，已有报道利用SPR检测固定蛋白的构象变化。然而，这一特性尚未被用于检测与固定蛋白受体结合的低分子量配体。在这里，我们证明了配体诱导的构象变化可以用来监测小分子与固定化麦芽糖结合蛋白和组织转谷氨酰胺酶的结合。配体与流体动力半径减小的受体结合产生折射率的净减小。在折射率的净正变化被观察到的受体，增加了流体动力半径。折射率的变化不能用分析物分子质量的增加来解释。根据反应的可逆性和SPR测定的平衡解离常数与报道的解离常数之间的相似性来判断，这些SPR反应是特异性受体-配体相互作用的结果。此外，这项技术被证明在检测小分子面板中的特定配体方面是有效的。这种SPR方法不需要改变广泛使用的和商用的仪器，但可以直接检测非常小的分子，如钙离子（40 Da）。利用受体构象检测低分子量分析物在小分子药物文库的高通量筛选和生物传感器的开发中具有潜在的应用前景。
Small molecules are difficult to directly detect using commercially available surface plasmon resonance (SPR) instruments. Ibis is because low molecular weight compounds do not have sufficient mass to cause a measurable change in refractive index. Refractive index is sensitive, however, to other properties besides the mass of the analyte. Recently the detection of substantial conformational changes for immobilized proteins using SPR has been reported. However, this property has not yet been exploited for the detection of low molecular weight ligand binding to immobilized protein receptors. Here we demonstrate that ligand-induced conformational changes can be used to monitor the binding of small molecules to immobilized maltose-binding protein and tissue transglutaminase. Ligand binding to a receptor that decreases in hydrodynamic radius yielded a net decrease in refractive index. A net positive change in refractive index was observed for a receptor that increases in hydrodynamic radius. Refractive index changes could not be explained by addition of analyte molecular mass to the surface. These SPR responses were a result of specific receptor-ligand interactions, as judged by the reversibility of the response and the similarities between the SPR-determined equilibrium dissociation constants and reported dissociation constants. Additionally, this technique proved to be effective at detecting specific ligands from a panel of small molecules. This SPR method required no alterations in widely used and commercially available instrumentation yet allowed direct detection of very small molecules such as calcium ions (40 Da). Use of receptor conformation to detect low molecular weight analytes has potential applications in the high-throughput screening of small molecule drug libraries and the development of biosensors.