Simple and Efficient Strategy for Site-Specific Dual Labeling of Proteins for Single-Molecule Fluorescence Resonance Energy Transfer Analysis

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• 作者：Jihyo Kim ; Moon-Hyeong Seo ; Sangsik Lee ; Kyukwang Cho ; Aerin Yang ; Kyunghwa Woo ; Hak-Sung Kim ; Hee-Sung Park
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：February 5, 2013
• 年：2013
• 卷：85
• 期：3
• 页码：1468-1474
• 全文大小：367K
• 年卷期：v.85,no.3(February 5, 2013)
• ISSN：1520-6882

文摘

Analysis of protein dynamics using single-molecule fluorescence resonance energy transfer (smFRET) is widely used to understand the structure and function of proteins. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. Here we present a general and facile method for site-specific dual labeling of proteins by incorporating two different, readily available, unnatural amino acids (p-acetylphenylalanine and alkynyllysine) for smFRET. We used newly evolved alkynyllysine-specific aminoacyl-tRNA synthetase/tRNA_UCA and p-acetylphenylalanyl-tRNA synthetase/tRNA_CUA. The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics.