Abstract:Histone lysine residues can be mono-, di-, or trimethylated. These posttranslational modifications regulate the affinity of effector proteins and may also impact chromatin structure independent of their role as adaptors. In order to study histone lysine methylation, particularly in the context of chromatin, we have developed a chemical approach to install analogs of methyl lysine into recombinant proteins. This approach allows for the rapid generation of large quantities of histones in which the site and degre… Show more
“…Methyl-lysine Histone Octamer Preparation-Lysine 27 of histone H3 was mutated to a cysteine by site-directed mutagenesis of Xenopus H3.1 bearing a C110A mutation, expressed and purified from Escherichia coli inclusion bodies, and subjected to chemical alkylation by (2-bromoethyl)trimethylammonium bromide before assembly into histone octamers (22,23). Modification of the histone tail was verified by using nano-spray mass spectrometry.…”
“…Methyl-lysine Histone Octamer Preparation-Lysine 27 of histone H3 was mutated to a cysteine by site-directed mutagenesis of Xenopus H3.1 bearing a C110A mutation, expressed and purified from Escherichia coli inclusion bodies, and subjected to chemical alkylation by (2-bromoethyl)trimethylammonium bromide before assembly into histone octamers (22,23). Modification of the histone tail was verified by using nano-spray mass spectrometry.…”
“…2a) 26,27 . More recently, transition metalcatalysed modifications of cysteine have been reported, such as rhodium-catalysed reaction with diazo compounds, although potential side reactions with tryptophan have been noted as a limitation 28 .…”
Section: Modifications Of Natural Amino Acidsmentioning
“…We previously reported that histones containing methyl-lysine analogues (MLA) [6] are compatible in biochemical reactions mediated by many histone methyltransferases, including Dot1L, HYPB, Suvar4-20 and PrSet7 [7]. We then extended this study to G9a.…”
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