Detection of conformational changes in an immobilized protein using surface plasmon resonance.
Detection of conformational changes in an immobilized protein using surface plasmon resonance.
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DOI：
10.1021/ac9713666
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发表时间：
1998-04
影响因子：
7.4
通讯作者：
H. Sota;Yukio Hasegawa;Masahiro Iwakura
中科院分区：
文献类型：
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作者：
H. Sota;Yukio Hasegawa;Masahiro Iwakura
利用表面等离子共振 (SPR)，我们开发了检测固定化蛋白质构象变化的新方法。基因改造的大肠杆菌二氢叶酸还原酶 (DHFR-ASC) 通过工程蛋白 C 末端的二硫键连接到覆盖 SPR 生物传感器传感器表面的羧甲基葡聚糖基质层上。与缺乏固定蛋白质的参考表面相比，DHFR-ASC 固定表面对酸处理表现出更大的响应。束缚蛋白的 SPR 信号和溶液中 DHFR-ASC 的摩尔椭圆率对 pH 变化的响应类似，这与 SPR 信号的变化反映酸变性过程中发生的构象变化的解释一致。在 SPR 信号和椭圆度变化之间观察到的 pH 值变化可能反映了表面 pH 值与本体 pH 值之间的差异。束缚蛋白传感器表面对于使用 pH 值范围为 0.12-7.80 的溶液进行重复酸处理是稳定的，并且产生了可重复的测量结果。这是使用 SPR 生物传感器检测固定化蛋白质构象变化的首次演示。该技术具有开发响应蛋白质构象变化的新型传感器和/或开关装置的潜力。
Utilizing surface plasmon resonance (SPR), we have developed novel methodology for the detection of conformational change(s) in immobilized proteins. A genetically altered E. coli dihydrofolate reductase (DHFR-ASC) was attached to a carboxymethyldextran matrix layer covering the sensor surface of an SPR biosensor through a disulfide linkage at the engineered protein's C-terminus. The DHFR-ASC-immobilized surface exhibited a larger response to acid treatment than reference surfaces lacking immobilized proteins. The SPR signal of the tethered protein and the molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR signal reflect conformational changes occurring during acid denaturation. A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH. The tethered protein sensor surface was stable to repeated acid treatment using solutions in the pH range of 0.12-7.80 and yielded reproducible measurements. This is the first demonstration of detection of conformational changes in an immobilized protein using an SPR biosensor. This technique has potential for developing novel sensors and/or switching devices in response to protein conformational changes.