化学
费斯特共振能量转移
氨基酸
标签
翻译(生物学)
计算生物学
荧光
生物化学
生物物理学
组合化学
基因
信使核糖核酸
物理
量子力学
生物
作者
Kaihang Wang,Amit Sachdeva,Daniel J. Cox,Nabil M. Wilf,Kathrin Lang,Stephen Wallace,Ryan A. Mehl,Jason W. Chin
出处
期刊:Nature Chemistry
[Springer Nature]
日期:2014-04-20
卷期号:6 (5): 393-403
被引量:231
摘要
The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer. A series of quadruplet decoding tRNAs has been developed to form an optimized orthogonal translation system. These tRNAs enable efficient, site-specific incorporation of multiple unnatural amino acids into a protein, with a substantial increase in yield over previous methods. The amino acids are then used to site-specifically label a protein with a pair of fluorophores, enabling studies of the protein's dynamics.
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