Toward D-peptide biosynthesis: Elongation Factor P enables ribosomal incorporation of consecutive D-amino acids

Abstract

To maintain stereospecific biochemistry in cells, living organisms have evolved mechanisms to exclude D-amino acids ( D AA) in their protein synthesis machinery, which also limits our exploration of the realm of mirror-image molecules. Here, we show that high affinity between EF-Tu and aminoacyl-tRNA promotes D-amino acid incorporation. More strikingly, Elongation Factor P efficiently resolves peptidyl transferase stalling between two consecutive D-amino acids, and hence enables the translation of D-peptides.Life is an anti-entropic phenomenon with two mutually-reinforcing characters: homochirality and stereospecific catalysis. The exclusive presence of L-amino acids in proteins of the living world is a prominent example of this. However, D-amino acid containing peptides (DAACP) are still present in microbial, fungal and amphibian secretions, and often carry interesting bioactivities 1 . In nature, these molecules are made through non-ribosomal pathways, such as non-ribosomal peptide synthesis or post-translational modification, e.g. epimerization. DAACPs have been shown to have prolonged half lives in serum and resistance toward proteases without immunogenicity 2 , which is a desirable property in therapeutic reagents. Therefore, by gradually rewiring the present bio-machineries to overcome natural D AA barrier, we aim to build a bridge leading us to the space of mirror-image biomolecules, where they can serve as new tools for the biotech and pharmaceutical industry.Core protein translation machinery exhibits significant discrimination of D AAs from L-amino acids ( L AAs) at three steps 3 : aminoacylation of tRNAs by aminoacyl-tRNA synthetases (aaRSes), formation of ternary complexes with EF-Tu-GTP, and peptide-bond formation catalyzed by the ribosome (Figure 1a). In order to study these discriminations without the interference from D-amino acid oxidase and D-aminoacyl-tRNA deacylase 4,5 , we use a purified E. coli protein synthesis system (PURE) 6 , along with chemically acylated D-aminoacyl-tRNA 7 , as a model system for engineering D AA-tolerant translation machinery. We adapted the amber codon read through assay described in Fujino et al. 8 with slight modifications to assess D AA incorporation during elongation (Figure 1b). In brief, mRNA templates encoding an N-terminus FLAG epitope and a C-terminus stretch of artificial peptide composed of six different amino peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. Incorporation could possibly be improved by additional EF-Tu and higher concentrations of charged tRNAs in the in vitro translation system, but we had earlier found that this only achieved a moderate improvement in L Ala 11 . We therefore thought to compensate for the disadvantage of low-incorporating L/D AAs by using a tRNA body that binds very efficiently to EF-Tu, noting that the K D s of tRNA bodies to EF-Tu-GTP span 600-fold from the weakest to the strongest 9 . Therefore, we chose four tRNA bodies (Glu2, Thr2, Gly2 and Ala2, sequences provided in Sup...

Fundamental Clock of Biological Aging: Convergence of Molecular, Neurodegenerative, Cognitive, and Psychiatric Pathways: Non-Equilibrium Thermodynamics Meet Psychology

“…Small amount D-AAs in an organism signify the evolutionary selected delicate balance between two isomers, suggesting that any disturbance in this balance can be harmful. It is notable that D-AA-containing peptides and proteins demonstrate two distinct features: (1) resistance toward proteases and association with prolonged halflives (2) [60,62]. Biochirality, evident in the pathways of enzymatic and spontaneous racemization of AAs and PTMs of proteins, is the emerging as a critical and yet experimentally and theoretically challenging topic.…”

Continuous‐Variable Quantum Key Distribution with Gaussian Modulation—The Theory of Practical Implementations

“…By now, many experimental demonstrations underlined the feasibility of CV-QKD with coherent states. [14][15][16][17][18][19][20][21][22][23][24][25] The experimental performance of these works has to be evaluated in the context of the various settings and assumptions that the respective authors applied and are therefore difficult to compare. After all, a CV-QKD setup is primarily appraised by the secure-key rate that can be achieved over a given transmission distance.…”

“…For example, a few papers so far successfully demonstrated longdistance CV-QKD with a non-zero key over a channel length of 80-100 km. [17,21] On the other hand, in these implementations, the local oscillator (a strong laser signal required to measure the quantum signals) is transmitted to the receiver over the quantum channel and is therefore accessible to possible manipulations by an eavesdropper-a deficiency which opens a number of severe security loopholes. [26][27][28][29][30][31][32][33] This is why recent works are focusing on different methods to avoid the transmission of the local oscillator and to generate it at the receiver lab instead (often referred to as "true local oscillator" or "local local oscillator" [LLO]).…”

From Designing the Molecules of Life to Designing Life: Future Applications Derived from Advances in DNA Technologies

“…[64] Theobservation that elongation factor EF-P alleviates ribosome peptidyl transferase stalling at polyproline stretches on ag rowing ribosomal peptide chain [65] motivated our lab to use EF-P to alleviate stalling that occurs between two consecutive Damino acids. [66] Recent work has demonstrated consecutive elongation of D-AAs in translation using complementary strategies. [67] Theability of fully D-AA-containing enzymes to act on opposite chirality has been demonstrated using aD -AA polymerase on an L-DNAt emplate,w hich rendered af unctionally active l-DNAzyme.…”