Collauto Alberto, DeBerg Hannah A, Kaufmann Royi, Zagotta William N, Stoll Stefan, Goldfarb Daniella
Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 76100, Israel.
Phys Chem Chem Phys. 2017 Jun 14;19(23):15324-15334. doi: 10.1039/c7cp01925d.
Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (μRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNBD) of the HCN2 ion channel upon binding of the ligand 3',5'-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC + L ⇌ AO + L ⇌ BO ⇌ BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved μRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.
配体结合可诱导蛋白质发生显著的构象变化。这一过程的机制将与配体结合事件相关的平衡和构象变化联系起来。在这里，我们表明，通过将W波段双电子-电子共振（DEER）光谱与微流控快速冷冻淬灭（μRFQ）相结合，可以解析这些过程，并获得平衡常数和反应速率。我们研究了HCN2离子通道的硝基氧标记的、分离的羧基末端环核苷酸结合结构域（CNBD）在配体3',5'-环磷酸腺苷（cAMP）结合时的构象转变。使用基于模型的全局分析，μRFQ DEER实验的时间分辨数据直接提供了开放和封闭构象的分数群体随时间的变化情况。我们使用四态模型对蛋白质中配体诱导的构象变化进行建模：无配体/开放（AO）、无配体/封闭（AC）、结合/开放（BO）、结合/封闭（BC）。这些状态根据AC + L ⇌ AO + L ⇌ BO ⇌ BC相互转化。通过分析平衡时封闭和开放构象相对贡献的浓度依赖性，我们估计了两个构象平衡的平衡常数以及开放态配体解离常数。对时间分辨的μRFQ DEER数据的分析给出了配体结合和解离的内在速率以及构象变化速率的估计值。这表明DEER可以定量解析配体结合的热力学和动力学以及相关的构象变化。
