N-Terminal Protein Modification Using Simple Aminoacyl Transferase Substrates

Abstract

Methods for synthetically manipulating protein structure enable greater flexibility in the study of protein function. Previous characterization of the E. coli aminoacyl tRNA transferase (AaT) has shown that it can modify the N-terminus of a protein with an amino acid from a tRNA or a synthetic oligonucleotide donor. Here, we demonstrate that AaT can efficiently use a minimal adenosine substrate, which can be synthesized in one to two steps from readily available starting materials. We have characterized the enzymatic activity of AaT with aminoacyl adenosyl donors and found that reaction products do not inhibit AaT. The use of adenosyl donors removes the substrate limitations imposed by the use of synthetases for tRNA charging and avoids the complex synthesis of an oligonucleotide donor. Thus, our AaT donors increase the potential substrate scope and reaction scale for Nterminal protein modification under conditions that maintain folding.

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

“…S3). Comparing the apparent K i (uncharged tRNA Leu = 31 μM and uncharged tRNA Phe = 25 μM) with the apparent K m (Leu-tRNA Leu = 2 μM and PhetRNA Phe = 3 μM in this study and rA-Phe = 124 μM [Wagner et al 2011]) confirms that the recognition of aa-tRNA substrate by L/F transferase is through both the amino acid moiety and the tRNA body. The amino acid moiety contributes to an approximately eightfold difference (comparing Phe-tRNA Phe with uncharged tRNA Phe ), while the tRNA body contributes to an ∼40-fold difference (comparing Phe-tRNA Phe with rA-Phe) in affinity, further confirming that the tRNA body does contribute significantly to recognition.…”

“…The current tRNA recognition model does not explain this isoacceptor preference. A comparison with the reported apparent K m values for rAPhe (124 μM) and Phe-tRNA Phe (1.56 μM), suggests that the tRNA body contributes to the L/F transferase recognition significantly (Rao and Kaji 1974;Abramochkin and Shrader 1996;Wagner et al 2011). In vitro assays with mutant tRNAs suggest that the anticodon and variable loop are not the basis for specificity (Abramochkin and Shrader 1996).…”

“…A model for L/F transferase tRNA recognition has been proposed based on biochemical and structural data (Leibowitz and Soffer 1971;Abramochkin and Shrader 1996;Suto et al 2006;Watanabe et al 2007). In vitro studies with misacylated tRNAs (Leibowitz and Soffer 1971;Abramochkin and Shrader 1996) and minimalistic adenosine esters of natural and unnatural amino acids (3 ′ rA-aa) (Wagner et al 2011) are sufficient for aminoacyl transfer. This suggests that the major determinant of tRNA recognition by L/F transferase is the 3 ′ terminal adenosine and the aminoacyl moiety of an aa-tRNA (Leibowitz and Soffer 1971;Abramochkin and Shrader 1996).…”

A peptide ligase and the ribosome cooperate to synthesize the peptide pheganomycin

“…Furthermore, PGM1 may be useful for modifying the N-terminus of proteins of interest, since there are many reports demonstrating such trials by chemical and enzyme methods for a diverse range of aims [30][31][32][33] .…”

Expressed Protein Ligation at Methionine: N‐Terminal Attachment of Homocysteine, Ligation, and Masking

“…[11] Previous work from Tirrell and Sisido demonstrated that AaT could transfer unnatural amino acids from Zaa-tRNAs produced either through semi-synthesis or by the activity of a modified aminoacyl tRNA synthetase (RS). [12] These two strategies are complementary.…”