Multicolor protein FRET with tryptophan, selective coumarin-cysteine labeling, and genetic acridonylalanine encoding

被引:20
|
作者
Ferrie, John J. [1 ]
Ieda, Naoya [1 ,2 ]
Haney, Conor M. [1 ]
Walters, Christopher R. [1 ]
Sungwienwong, Itthipol [1 ]
Yoon, Jimin [1 ]
Petersson, E. James [1 ]
机构
[1] Univ Penn, Dept Chem, 213 South 34th St, Philadelphia, PA 19104 USA
[2] Nagoya City Univ, Grad Sch Pharmaceut Sci, Dept Organ & Med Chem, Mizuho Ku, 3-1 Tanabedori, Nagoya, Aichi 4678603, Japan
基金
美国国家卫生研究院; 日本学术振兴会;
关键词
RESONANCE ENERGY-TRANSFER; AMINO-ACID; ALPHA-SYNUCLEIN; PEPTIDE COMPLEXES; CALMODULIN; DISSOCIATION; LANDSCAPE; STABILITY; CHANNEL; CELLS;
D O I
10.1039/c7cc05492k
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Site-specific fluorescence probes can be used to measure distances within proteins when used as part of a Forster resonance energy transfer (FRET) pair. Here we report the synthesis of a coumarin maleimide (Mcm-Mal) that is fluorogenic upon reaction with cysteine. We demonstrate that cysteine, acridonylalanine (Acd) double mutant proteins can be produced by unnatural amino acid mutagenesis and reacted with Mcm-Mal to generate Mcm/Acd labeled proteins for FRET studies. The Mcm/Acd FRET pair is minimally-perturbing, easy to install, and well-suited to studying protein distances in the 15-40 angstrom range. Furthermore, Mcm/Acd labeling can be combined with tryptophan fluorescence in three color FRET to monitor multiple interactions in one experiment.
引用
收藏
页码:11072 / 11075
页数:4
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