Genetic incorporation of a metal-ion chelating amino acid into proteins as a biophysical probe.

Genetic incorporation of a metal-ion chelating amino acid into proteins as a biophysical probe.
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DOI:
10.1021/ja808340b
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发表时间:
2009-02-25
影响因子:
15
通讯作者:
Wang F
Wang F
中科院分区:
化学1区
文献类型:
--
作者:
Lee HS;Spraggon G;Schultz PG;Wang F

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在大肠杆菌E.大肠杆菌通过琥珀无义密码子和相应的正交tRNA/氨酰-tRNA合成酶对。将氨基酸掺入TM 0665蛋白中,并将突变蛋白与Zn 2+共结晶以通过SAD定相确定结构。该结构显示出与HQ-Ala残基结合的重金属的高占有率,并且重金属提供了优异的定相能力来确定结构。这种方法也有利于从头设计具有新结构和功能的金属蛋白,包括荧光传感器。
A metal-ion chelating amino acid, (8-hydroxyquinolin-3-yl)alanine, was genetically encoded inE. coliby an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. The amino acid was incorporated into TM0665 protein, and the mutant protein was cocrystallized with Zn2+to determine the structure by SAD phasing. The structure showed a high occupancy of the heavy metal bound to the HQ-Ala residue, and the heavy metal provided excellent phasing power to determine the structure. This method also facilitates the de novo design of metalloproteins with novel structures and functions, including fluorescent sensors.
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