Department of Biochemistry & Cellular and Molecular Biology, College of Arts and Sciences, University of Tennessee, 1311 Cumberland Avenue, Knoxville, TN 37932, USA.
Department of Chemistry, College of Liberal Arts and Sciences, University of Florida, 126 Sisler Hall, Gainesville, FL 32611, USA.
Structure. 2022 Mar 3;30(3):329-337.e5. doi: 10.1016/j.str.2021.11.005. Epub 2021 Dec 10.
A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A adenosine receptor (AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound AAR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available AAR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available AAR structures.
更完整的蛋白质能量景观描述包括确定不同功能相关构象状态以及确定连接它们的途径。我们使用全内反射荧光（TIRF）成像技术在单分子水平上研究了人类 A 腺苷受体（AAR）的构象动力学，AAR 是一种 A 类 G 蛋白偶联受体（GPCR）。在激动剂结合的 AAR 中，观察到三种不同荧光发射状态之间的缓慢、可逆构象交换。这些状态之间的转变主要按特定顺序发生，无活性和类似活性构象之间的交换通过中间状态进行。基于具有可用 AAR 结构的分子动力学模拟得出的模型可以合理地解释最高和最低发射状态的相对荧光发射强度，但不能解释过渡态。这表明，目前在可用的 AAR 结构中无法观察到实现激活所需的功能关键中间状态。
