We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic non-canonical amino acid L-β-(thieno[3,2-b]pyrrolyl)-alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp → [3,2] Tpa substitutions in their proteomes in response to the 20,899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition are capable of robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.
Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in an umber of theoretical studies.E xperimental evidence for such pathways, however,i ss parse because site-selective injection of vibrational energy into aprotein, that is,localized heating,isrequired for their investigation. Here,wesolved this problem by the site-specific incorporation of the non-canonical amino acid b-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kashasr ule,A zAla undergoes ultrafast internal conversion and heating after S 1 excitation while upon S 2 excitation, it serves as af luorescent label. We equipped PDZ3, ap rotein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions.W eindeed observed VET from the incorporation sites in the protein to ab ound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in aw ide range of proteins. Dr.J .Jaric Present address:H ospira Zagreb d.o.o.,aPfizer company Prudnicka cesta 60, 10291 Prigorje Brdovecko (Croatia) [ + + ]T hese authors contributed equally to this work. Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.
Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4-dihydroxyphenylalanine (DOPA)-rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl-transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho-nitrobenzyl DOPA (ONB-DOPA). The engineered ONB-DOPARS enables in vivo production of MAP type 5 site-specifically equipped with multiple instances of ONB-DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives.
The acidity of N-acyl amino acids is dependent upon the rotameric state of the amide bond. In this work we systematically investigated the acidity difference of the rotamers (Delta pK(a)) in the frames of various acetylated amino acids. Our results indicated a mutual interaction of two carbonyl groups of an attractive type. We observed conservative Delta pK(a)s for acyclic amino acids (2.2-3.0 kJ mol(-1)), whereas in the case of alicyclic amino acids, the experimental values revealed a strong dependency on the structural context (1.5-4.4 kJ mol(-1)). In homologous amino acids (alpha-, beta-, gamma-, etc.), the strength of the attraction decays in an exponential fashion. Furthermore, the interaction can accumulate through a chain of amide bonds in a cascade fashion, as demonstrated by an Ac-Pro-Pro dipeptide. As a result, we demonstrate that Delta pK(a) is an experimental parameter to estimate increments in the carbonyl-carbonyl alignment, as determined by the amino acid or peptidyl context. This parameter is also important in understanding the roles of amino acids in both protein folding and translation in biological systems as well as their evolutionary appearance in the genetic code.DFG, EXC 314, Unifying Concepts in Catalysi
Albicidin is ar ecently described natural product that strongly inhibits bacterial DNA gyrase. The pronounced activity,particularly against Gram-negative bacteria, turns it into ap romising lead structure for an antibacterial drug. Hence, structure-activity relationship studies are key for the in-depth understanding of structuralf eatures/moieties affectingg yrase inhibition, antibacteriala ctivity ando vercomingr esistance. The 27 newly synthesized albicidinsg ive profound insights into possibilities for variations of the C-terminus.F urthermore, in the present study,anovel derivative has been identified as overcoming resistance posed by the Klebsiella-protease AlbD. Structural modifications include, for example,a zahistidine replacing the previous instable cyanoalanine as the central amino acid, as well as at riazole amide bond isostere between building blocks Da nd E.
Vibrational energy transfer (VET) is essential for protein function. It is responsible for efficient energy dissipation in reaction sites, and has been linked to pathways of allosteric communication. While it is understood that VET occurs via backbone as well as via non-covalent contacts, little is known about the competition of these two transport channels, which determines the VET pathways. To tackle this problem, we equipped the β-hairpin fold of a tryptophan zipper with pairs of non-canonical amino acids, one serving as a VET injector and one as a VET sensor in a femtosecond pump probe experiment. Accompanying extensive non-equilibrium molecular dynamics simulations combined with a master equation analysis unravel the VET pathways. Our joint experimental/computational endeavor reveals the efficiency of backbone vs. contact transport, showing that even if cutting short backbone stretches of only 3 to 4 amino acids in a protein, hydrogen bonds are the dominant VET pathway.
Genetic code engineering that enables reassignment of genetic codons to non-canonical
amino acids (ncAAs) is a powerful strategy for enhancing ribosomally synthesized
peptides and proteins with functions not commonly found in Nature. Here we report
the expression of a ribosomally synthesized and post-translationally modified
peptide (RiPP), the 32-mer lantibiotic lichenicidin with a canonical tryptophan
(Trp) residue replaced by the ncAA
L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) which does
not sustain cell growth in the culture. We have demonstrated that cellular toxicity
of [3,2]Tpa for the production of the new-to-nature bioactive congener of
lichenicidin in the host Escherichia coli can be alleviated by using an
evolutionarily adapted host strain MT21 which not only tolerates [3,2]Tpa but also
uses it as a proteome-wide synthetic building block. This work underscores the
feasibility of the biocontainment concept and establishes a general framework for
design and large scale production of RiPPs with evolutionarily adapted host
strains.
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