一种基于荧光共振能量转移的组蛋白甲基转移酶检测方法。

A fluorescence resonance energy transfer-based method for histone methyltransferases.

作者信息

Devkota Kanchan, Lohse Brian, Jakobsen Camilla Nyby, Berthelsen Jens, Clausen Rasmus Prætorius

机构信息

Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark; NNF Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark.

Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark.

出版信息

Anal Biochem. 2015 May 1;476:78-80. doi: 10.1016/j.ab.2015.02.012. Epub 2015 Feb 19.

DOI:10.1016/j.ab.2015.02.012

PMID:25703602

Abstract

A simple dye-quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye and a quencher, respectively. When lysine-9 residues in the peptides were methylated, they were protected from cleavage by endoproteinase-EndoLysC, whereas unmethylated peptides were cleaved, resulting in an increase in fluorescent intensity.

摘要

开发了一种基于简单染料-猝灭剂荧光共振能量转移（FRET）的甲基转移酶检测方法，并用于测定EHMT1和EHMT2的动力学参数及抑制活性。模拟截短组蛋白H3尾巴的肽段在两端分别用一种染料和一种猝灭剂进行功能化修饰。当肽段中的赖氨酸-9残基被甲基化时，它们受到内肽酶-EndoLysC切割的保护，而未甲基化的肽段则被切割，导致荧光强度增加。