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.

中文翻译：

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通过压力位移测定法引起的变性剂或配体诱导的蛋白质体积变化

蛋白质-配体结合的完整热力学描述包括与压力和温度相关的参数。结合配体后蛋白质体积和可压缩性的变化是与压力相关的参数，由于缺乏常规测定方法，这些参数经常被忽略。荧光压力位移测定 (FPSA) 基于压力诱导的蛋白质展开及其通过配体的稳定性，并提供了一种确定蛋白质-配体结合体积的通用方法。大多数蛋白质和蛋白质-配体复合物的展开需要极高的压力。因此，盐酸胍 (GdmHCl) 被用作蛋白质去稳定剂。我们确定 GdmHCl 在不同的途径中展开碳酸酐酶同种型，但去稳定效应在特定浓度范围内是线性的。我们为 FPSA 实验开发了一个概念，其中配体和 GdmHCl 的浓度都是不同的。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的设备无法达到的压力，这是不可能的。