An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET.
An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET.
复制标题
一种用于利用时间分辨过渡金属离子荧光共振能量转移（FRET）测量构象分布的改良型荧光非经典氨基酸
DOI：
10.7554/elife.70236
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发表时间：
2021-10-08
期刊：
影响因子：
7.7
通讯作者：
Gordon SE
中科院分区：
文献类型：
--
作者：
Zagotta WN;Sim BS;Nhim AK;Raza MM;Evans EG;Venkatesh Y;Jones CM;Mehl RA;Petersson EJ;Gordon SE
随着最近高分辨率蛋白质结构的爆发，生物学的下一个前沿领域之一是阐明在变化的条件下调节蛋白质的构象重排以满足细胞需要的机制。严格测量蛋白质的能量学和动力学需要开发新的方法来解决结构异质性和构象分布。我们之前已经开发了稳态过渡金属离子荧光共振能量转移（tmFRET）方法，使用荧光非规范氨基酸供体（Anap）和过渡金属离子受体来探测可溶性和膜蛋白的构象重排。在这里，我们证明了荧光非规范氨基酸具有优越的光物理性质，扩展了其作为tmFRET供体的效用。使用在哺乳动物细胞中表达的麦糖结合蛋白（MBP）作为模型系统，我们发现在稳态tmFRET实验中，add与Anap相当，并且其长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在载脂蛋白和全息构象态上的异质性差异，并精确量化了亚饱和麦芽糖浓度下载脂蛋白和全息构象态的分布。我们的新方法使用add进行时间分辨tmFRET，为在接近自然条件下测量可溶性和膜蛋白的构象重排的能量学奠定了基础。
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.