Improving engineering of ribosomal peptidomimetic synthesis based upon fast kinetics analyses

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Jinfan Wang, Ka-Weng Ieong, Michael Y. Pavlov, Marek Kwiatkowski, Mns Ehrenberg, Anthony C. Forster

Uppsala University, Sweden

The ribosomal incorporation of single and multiple unnatural amino acids (AAs) for fabrication of redesigned peptides and biological systems is generally limited by low incorporation efficiencies. We used our purified translation system and quench-flow techniques to study the kinetics of translation with unnatural AA-tRNAs prepared via the standard aminoacyl-pdCpA chemoenzymatic ligation method [1,2]. Single incorporation of allyl-glycine (aG) into peptide had a similar rate to Phe [3]. However, the binding affinity of aG-tRNA to EF-Tu was lower [3], indicating that the low incorporation efficiency of aG [4] was caused by inefficient delivery. Furthermore, in kinetic studies of five consecutive incorporations with aG, the processivity was lower than with natural Ala-tRNA(Ala). This is in a line with our earlier observation [4] that the deoxyC introduced to the tRNA body in the chemoenzymatic ligation method decreased the yield. Peptidyl-tRNA hydrolase assays indicated that the lower processivities were caused by drop-off of intermediate-length peptides. Full processivity could not be recovered by increasing EF-Tu concentration, indicating that the delivery of the AA-tRNA was not limiting in the case of multiple adjacent unnatural AA incorporations. We conclude that the incorporation efficiencies of single non-N-alkylated unnatural AAs can be improved by increasing EF-Tu concentration, while incorporations of multiple unnatural AAs might be improved using aminoacyl-pCpA instead of aminoacyl-pdCpA. References: 1. S A Robertson, C J Noren, S J Anthony-Cahill, M C Griffith, P G Schultz (1989) Nucleic Acids Res. 17,9649. 2. M Y Pavlov, R E Watts, Z Tan, V W Cornish, M Ehrenberg, A C Forster (2009) PNAS. 106(1), 50. 3. K-W Ieong, M Y Pavlov, M Kwiatkowski, A C Forster, M Ehrenberg (2012) JACS. 134, 17955. 4. R Gao, A C Forster (2010) FEBS Lett. 584(1), 99.