Abstract：
:Methods for installing natural and unnatural amino acids and their modifications into proteins in a benign and precise manner are highly sought-after in protein science. Here we describe a protocol for 'post-translational mutagenesis' that enables the programmed installation of protein side chains through the use of rapid, mild and operationally simple free-radical chemistry performed on recombinantly expressed and purified proteins. By introduction of protein dehydroalanine (Dha) residues (in this instance, from a unique cysteine residue introduced by site-directed mutagenesis) as free-radical trapping 'tags' for downstream modification, exquisite control over the site of subsequent modification is achieved. Using readily available alkyl halide precursors and simple borohydride salts, alkyl radicals can be generated in aqueous solution. These alkyl radicals react rapidly with protein-bound Dha residues to yield functionalized protein products with new carbon-carbon bonds. Once the Dha is installed, the introduction of the desired functionality is limited only by the requirement for polarity matching of the generated radical with the Dha 'acceptor', the solubility of the alkyl halide precursors in aqueous solution and the kinetics of the reaction itself. For example, methylated derivatives of lysine, arginine and glutamine are readily accessible. Furthermore, as the side chains are constructed chemically, many unnatural modifications can also be directly introduced as part of the side chain, including isotope reporters (19F, 13C) that can be used in biophysical experiments such as protein NMR. From a suitable cysteine mutant of the target protein, the entire procedure for this chemical post-translational mutation takes 2 d and is readily performed by nonchemists.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Wright TH,Davis BGdoi
10.1038/nprot.2017.087subject
Has Abstractpub_date
2017-10-01 00:00:00pages
2243-2250issue
10eissn
1754-2189issn
1750-2799pii
nprot.2017.087journal_volume
12pub_type
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