Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity
Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity
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DOI：
10.1073/pnas.0910421107
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发表时间：
2010-03-16
影响因子：
11.1
通讯作者：
Hellinga, Homme W.
中科院分区：
文献类型：
--
作者：
Isom, Daniel G.;Vardy, Eyal;Hellinga, Homme W.
天然状态和未折叠状态之间的吉布斯自由能差(“稳定性”)是蛋白质的基本特征之一。通过利用配体结合和稳定性之间的热力学联系，可以检测和量化蛋白质与小分子、核酸或其他蛋白质的相互作用。因此，蛋白质稳定性的测定可以提供生化功能的通用监测。然而，稳定性测量作为功能探针的使用并未得到充分利用，因为此类实验传统上需要大量蛋白质和特殊仪器。在这里，我们提出了定量半胱氨酸反应性(QCR)技术，仅使用皮摩尔量的材料和容易获得的实验室设备，就可以快速而准确地确定蛋白质的稳定性。我们证明QCR衍生的稳定性可以用来测量在广泛的配基浓度和亲和力范围内的配基结合。我们预计这项技术将在高通量蛋白质工程实验和功能基因组学中有广泛的应用。
The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics.