Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, 10257 Vilnius, Lithuania.
Phys Chem Chem Phys. 2022 Jul 21;24(28):17279-17288. doi: 10.1039/d2cp01046a.
A complete thermodynamic description of protein-ligand binding includes parameters related to pressure and temperature. The changes in the protein volume and compressibility upon binding a ligand are pressure-related parameters that are often neglected due to the lack of routine methods for their determination. Fluorescent pressure shift assay (FPSA) is based on pressure-induced protein unfolding and its stabilization by a ligand and offers a universal approach to determine protein-ligand binding volumes. Extremely high pressures are required to unfold most proteins and protein-ligand complexes. Thus, guanidinium hydrochloride (GdmHCl) is used as a protein-destabilizing agent. We determined that GdmHCl unfolds carbonic anhydrase isoforms in a different pathway, but the destabilization effect is linear in a particular concentration range. We developed a concept for the FPSA experiment, where both - the ligand and GdmHCl - concentrations are varied. This approach enabled us to determine protein-ligand binding volumes that otherwise would be impossible due to the equipment-unreachable pressures of protein unfolding.
完整的蛋白质-配体结合的热力学描述包括与压力和温度相关的参数。由于缺乏常规的测定方法，配体结合时蛋白质体积和压缩性的变化是与压力相关的参数，通常被忽略。荧光压力位移测定法（FPSA）基于压力诱导的蛋白质展开及其被配体稳定，并提供了一种通用的方法来确定蛋白质-配体结合体积。大多数蛋白质和蛋白质-配体复合物需要极高的压力才能展开。因此，盐酸胍（GdmHCl）被用作蛋白质变性剂。我们确定 GdmHCl 以不同的途径展开碳酸酐酶同工酶，但在特定浓度范围内，其失稳效应是线性的。我们提出了 FPSA 实验的概念，其中同时改变配体和 GdmHCl 的浓度。这种方法使我们能够确定蛋白质-配体结合体积，否则由于蛋白质展开所需的设备不可及的压力，这些体积是不可能确定的。
