Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Anal Biochem. 2013 Dec 1;443(1):52-7. doi: 10.1016/j.ab.2013.08.015. Epub 2013 Aug 29.
The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (≥60°C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples.
小分子配体的鉴定是药物开发的重要第一步，特别是针对没有内在活性的蛋白质的药物。为此，重要的是要有能够快速准确地测量大量潜在配体结合亲和力的技术。因为配体结合以依赖于浓度和结合亲和力的方式稳定蛋白质结构，所以结合引起的蛋白质稳定化效应的大小可用于鉴定和表征配体。例如，蛋白质变性温度（Tm 移）的变化已成为鉴定潜在配体的一种流行方法。然而，Tm 移不能轻易转化为结合亲和力，并且在变性温度（≥60°C）下获得的配体排序不一定与生理温度下的排序一致。另一种方法是使用化学变性，它可以在任何温度下进行。化学变性移位允许在非常宽的动态范围内（高微摩尔至亚纳摩尔）准确地确定结合亲和力，并且在结合改变解折叠转变的协同性的情况下。在本文中，我们开发了基本的分析方程，并提供了几个实验示例。
