Department of Physiology and Biophysics, University of Washington, Seattle, United States.
Department of Chemistry, University of Pennsylvania, Philadelphia, United States.
Elife. 2021 Oct 8;10:e70236. doi: 10.7554/eLife.70236.
With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.
随着高分辨率蛋白质结构的迅速发展，生物学的下一个前沿领域之一是阐明蛋白质构象重排的调控机制，以满足细胞在不断变化的条件下的需求。严格测量蛋白质的能量和动力学需要开发新的方法，这些方法可以解决结构异质性和构象分布问题。我们之前已经开发了使用荧光非天然氨基酸供体（Anap）和过渡金属离子受体的稳态过渡金属离子荧光共振能量转移（tmFRET）方法，以探测可溶性和膜蛋白中的构象重排。在这里，我们表明，荧光非天然氨基酸 Acd 具有优越的光物理性质，从而扩展了其作为 tmFRET 供体的用途。我们使用在哺乳动物细胞中表达的麦芽糖结合蛋白（MBP）作为模型系统，表明 Acd 在稳态 tmFRET 实验中与 Anap 相当，并且其长的单指数寿命更适合使用时间分辨 FRET 探测构象分布。这些实验揭示了 MBP 的 apo 和 holo 构象态的异质性差异，并在亚饱和麦芽糖浓度下对 apo 和 holo 构象态之间的分布进行了准确的定量。我们使用 Acd 进行时间分辨 tmFRET 的新方法为在近天然条件下测量可溶性和膜蛋白构象重排的能量奠定了基础。
