It has been known for decades that proteins undergo conformational changes in response to binding ligands. Such changes are usually accompanied by a loss of entropy by the protein, and thus conformational changes are integral to the thermodynamics of ligand association. Methods to detect these alterations are numerous; here, we focus on the sedimentation velocity (SV) mode of AUC, which has several advantages, including ease of use and rigorous data-selection criteria. In SV, it is assumed that conformational changes manifest primarily as differences in the sedimentation coefficient (the s-value). Two methods of determining s-value differences were assessed. The first method used the widely adopted c(s) distribution to gather statistics on the s-value differences to determine whether the observed changes were reliable. In the second method, a decades-old technique called “difference SV” was revived and updated to address its viability in this era of modern instrumentation. Both methods worked well to determine the extent of conformational changes to three model systems. Both simulations and experiments were used to explore the strengths and limitations of the methods. Finally, software incorporating these methodologies was produced.

几十年来，人们已经知道蛋白质会响应结合配体而发生构象变化。这种变化通常伴随着蛋白质的熵损失，因此构象变化是配体结合热力学的组成部分。检测这些变化的方法有很多；在这里，我们专注于 AUC 的沉降速度 (SV) 模式，它有几个优点，包括易于使用和严格的数据选择标准。在 SV 中，假设构象变化主要表现为沉降系数（s值）的差异。评估了确定s值差异的两种方法。第一种方法使用了广泛采用的c ( s) 分布以收集有关s值差异的统计信息，以确定观察到的变化是否可靠。在第二种方法中，一种名为“差异 SV”的几十年前的技术得到复兴和更新，以解决其在现代仪器时代的可行性。这两种方法都可以很好地确定三个模型系统的构象变化程度。模拟和实验都被用来探索这些方法的优点和局限性。最后，产生了包含这些方法的软件。