Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
ACS Chem Biol. 2013 Aug 16;8(8):1664-70. doi: 10.1021/cb400267m. Epub 2013 Jun 18.
Nε-Acryloyl-l-lysine, a noncanonical amino acid with an electron deficient olefin, is genetically encoded in Escherichia coli using a pyrrolysyl-tRNA synthetase mutant in coordination with tRNACUAPyl. The acrylamide moiety is stable in cells, whereas it is active enough to perform a diverse set of unique reactions for protein modifications in vitro. These reactions include 1,4-addition, radical polymerization, and 1,3-dipolar cycloaddition. We demonstrate that a protein incorporated with Nε-acryloyl-l-lysine is efficiently modified with thiol-containing nucleophiles at slightly alkali conditions, and the acrylamide moiety also allows rapid radical copolymerization of the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions, the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific fluorescent labeling of proteins incorporated with Nε-acryloyl-l-lysine both in vitro and in living cells. This critical development allows easy access to an array of modified proteins for applications where high specificity and reaction efficiency are needed.
Nε-丙烯酰基-L-赖氨酸是一种具有缺电子烯烃的非典型氨基酸，可使用带有突变吡咯赖氨酸-tRNA 合成酶的大肠杆菌进行基因编码，并与 tRNACUAPyl 协调。丙烯酰胺部分在细胞中稳定，但其活性足以在体外进行一系列独特的蛋白质修饰反应。这些反应包括 1,4-加成、自由基聚合和 1,3-偶极环加成。我们证明，在略碱性条件下，与含巯基亲核试剂反应，可有效修饰掺入 Nε-丙烯酰基-L-赖氨酸的蛋白质，丙烯酰胺部分还允许相同蛋白质在生理 pH 值下快速自由基共聚成聚丙烯酰胺水凝胶。在生理条件下，丙烯酰胺基官能团与二芳基腈亚胺快速发生 1,3-偶极环加成反应，显示出开启的荧光。我们利用这一观察结果，在体外和活细胞中展示了掺入 Nε-丙烯酰基-L-赖氨酸的蛋白质的位点特异性荧光标记。这一关键进展为需要高特异性和反应效率的应用提供了大量修饰蛋白质的简便途径。
