Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Biophys Chem. 2014 Feb;186:3-12. doi: 10.1016/j.bpc.2013.08.006. Epub 2013 Aug 31.
The binding of ligands and substrates to proteins has been extensively studied for many years and can be described, in its simplest form, by two limiting mechanisms: conformational selection and induced fit. Conformational selection involves the binding of ligand to a pre-existing sparsely-populated conformation of the free protein that is the same as that in the final protein-ligand complex. In the case of induced fit, the ligand binds to the major conformation of the free protein and only subsequent to binding undergoes a conformational change to the final protein-ligand complex. While these two mechanisms can be dissected and distinguished by transient kinetic measurements, direct direction, characterization and visualization of transient, sparsely-populated states of proteins are experimentally challenging. Unless trapped, sparsely-populated states are generally invisible to conventional structural and biophysical techniques, including crystallography and most NMR measurements. In this review we summarize some recent developments in the use of paramagnetic relaxation enhancement to directly study sparsely-populated states of proteins and illustrate the application of this approach to two proteins, maltose binding protein and calmodulin, both of which undergo large rigid body conformational rearrangements upon ligand binding from an open apo state to a closed ligand-bound holo state. We show that the apo state ensemble comprises a small population of partially-closed configurations that are similar but not identical to that of the holo state. These results highlight the complementarity and interplay of induced fit and conformational selection and suggest that the existence of partially-closed states in the absence of ligand facilitates the transition to the closed ligand-bound state.
多年来，配体和底物与蛋白质的结合已被广泛研究，可以用两种极限机制来简单描述：构象选择和诱导契合。构象选择涉及到配体与自由蛋白质的预先存在的、稀疏分布的构象的结合，这种构象与最终的蛋白质-配体复合物中的构象相同。在诱导契合的情况下，配体结合到自由蛋白质的主要构象上，并且仅在结合之后才经历到最终的蛋白质-配体复合物的构象变化。虽然这两种机制可以通过瞬态动力学测量来分离和区分，但对蛋白质的瞬态、稀疏分布状态的直接定向、特征描述和可视化在实验上具有挑战性。除非被捕获，否则稀疏分布的状态通常对常规结构和生物物理技术是不可见的，包括晶体学和大多数 NMR 测量。在这篇综述中，我们总结了使用顺磁松弛增强来直接研究蛋白质的稀疏分布状态的一些最新进展，并说明了这种方法在两种蛋白质（麦芽糖结合蛋白和钙调蛋白）中的应用，这两种蛋白质在结合配体后都会发生大的刚体构象重排，从开放的apo 状态转变为封闭的配体结合的 holo 状态。我们表明，apo 状态的集合由一小部分部分关闭的构象组成，这些构象与 holo 状态的构象相似但不完全相同。这些结果突出了诱导契合和构象选择的互补性和相互作用，并表明在没有配体的情况下部分关闭状态的存在促进了向封闭的配体结合状态的转变。
