A Versatile Approach for Site‐Specific Lysine Acylation in Proteins

Abstract

Using amber suppression in coordination with am utant pyrrolysyl-tRNAs ynthetase-tRNA Pyl pair,a zidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with ap hosphinothioester allows the convenient synthesis of aprotein with asite-specifically installed lysine acylation. By simply changing the phosphinothioester identity,any lysine acylation type could be introduced. Using this approach,wedemonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su-H4 tetramer as asubstrate,wefurther confirmed that Sirt5 is an active histone desuccinylase.L ysine succinylation is arecently identified post-translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.

Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains

“…This approach opens a potentially interesting biomedical application area for theses ncAAs. Notably, ncAA substrates 5, 9, 12, 15, 17, 20, and 25 have been incorporated in a residue specific manner (using auxotrophy-based methods) but never in a site-specific mode, besides 17 [ 48 , 63 , 64 , 65 , 66 , 67 ]. Substrate 9 contains a cyclopropane ring with properties closely resembling to those of an olefinic double bond, which could be exploited for a wide range of site-specific bioorthogonal protein conjugation reactions [ 68 ].…”

The Chemical Biology of Reversible Lysine Post-translational Modifications

“…This limitation has been overcome using a multistep approach, termed a ''tag-and-modify'' strategy (Chalker et al, 2011). In this fashion, a two-step introduction of K Succ was achieved, whereby azidonorleucine (AznL) was installed and converted into different acyl-lysines via traceless-Staudinger ligation reactions (Wang et al, 2017b). By switching the acyl phosphinothioester, theoretically this strategy can be applied to any Lys acylation (Figure 3D) (Wang et al, 2015b).…”

DNAzymes for Amine and Peptide Lysine Acylation

“…25,26 One such reaction is lysine (Lys) acylation, where Lys acetylation is critical for histones and in other contexts, [27][28][29][30] and many longer-chain Lys acylation PTMs [31][32][33] such as malonylation, 34,35 succinylation, 34,36 and glutarylation 37,38 have been discovered yet are poorly understood. 39 As an alternative to approaches that include introduction of Lys analogues, [40][41][42][43] nonsense codon suppression, [44][45][46][47][48][49] bottom-up ligation-based assembly strategies, [50][51][52] or enzymatic methods that typically require creation of a non-native protein by insertion of a specific target sequence, [53][54][55][56][57][58] DNAzymes are promising for top-down introduction of Lys acylation PTMs onto intact native proteins, [59][60][61][62][63][64][65] but only if DNAzymes can be identified with the fundamental catalytic ability of Lys acylation.…”