Abstract
Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and β-AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints.
Highlights
Synthesis of proteins with exotic building blocks, noncanonical amino acids, can be achieved in vivo and in vitro
The repertoire of noncanonical amino acids (ncAAs) that can be introduced into peptides and proteins in vitro is larger than in vivo, due to the fact that the aminoacyl (AA)-tRNA substrates can be generated by Flexizyme, an artificial ribozyme with no ncAA specificity [1]
The majority of ncAAs have not been present in the course of natural evolution and tRNA synthetases have not yet evolved to reject them; this fact is exemplified by the apparent flexibility of wild-type synthetases which can, in vitro, aminoacylate even N-methylated and β-amino acids, as well as α, α-disubstituted amino acids [7]
Summary
Synthesis of proteins with exotic building blocks, noncanonical amino acids (ncAAs), can be achieved in vivo and in vitro. NcAA-tRNAs have to be synthesized by aminoacyl-tRNA synthetases (aaRSs) which have been naturally optimized during evolution to stringently recognize only one, canonical amino acid [6]. The majority of ncAAs have not been present in the course of natural evolution and tRNA synthetases have not yet evolved to reject them; this fact is exemplified by the apparent flexibility of wild-type synthetases which can, in vitro, aminoacylate even N-methylated and β-amino acids, as well as α, α-disubstituted amino acids [7]. An ncAA is aminoacylated by a wild-type aaRS and introduced in a residue-specific manner; the matching natural amino acid concentration is kept low by the use of a cAA-auxotrophic strain, while the ncAA is added to the growth medium. It is considered that aaRSs stabilize the transition state of both steps of aminoacylation [19], whicIht,isfocronthseidperuerdpothsaets aoafRaSasRsStaebnilgizineetehreintrga,nmsitaiyoncostnaftoeuonfdboththe svteispusaol finamspiencotaiocnylaotfiothne[1a9c]t,ivwehsicithe, faonrdththeepruorlpesosoefsinodf iavaiRduSaelnrgeisnideeureisngw,imthainy icto. nTfhouusn, dalthoeuvgishusatlruincstupreecstiofnaoafRtSh·eAaActaivnedsaiateRaSn·ndcAthAe rcoolmespolef xinedsivreidvueal rseisgindiufiecsawntitdhinffeitr.eTnhceuss,ianltthhoeughhydstrrougcetunrebsoonfdainaRgSp·AatAtearnds aanRdS·sntecAricAicnotmerpacletxioens rbeevtewaelesnignwifiilcda-ntytpdeiffaearRenSc·AesAinatnhde hayadRrSo·ngecnAbAoncodminpglpexaettser[n20s]a,nsdepstaerraitciningtetrhaectsiopnecbifeitcwreoelne wofiladc-tiyvpeeasiatReSr·eAsiAduaensdinaaiRniSti·nalcAsuAbsctoramteplbeixnedsin[2g0a],nsdepsatarbatiliinzgattihoensopfetchifiectrraonlesiotifoancstitvaete-srietemraeisnids uchesalilneningitniagl. sSuobpshtirsattiecabtienddianngaalynsdisstaanbdilizraetdioesnigonf tohfe trhaensaictitoivnestsaite rmemayainthsecrheafollreengreinqgu.irSeop>h1i5striceastidedueasnatolysbise amnudtaregdeensiizgendoafrtohuenadctitvheesaitcetimveaysittheearelofonree, rienquoirrdee>r1t5orcerseidauteesatosubpeemriourta, gnecnAizAe-dspaerociufnicdatahReSac[t2i1v]e. sCitoenacloomneit,aintolyr,dmerotroecsrterautcetaursaulpdeartioarm, nacyAbAe-snpeeecdifiecdataoRaSid[2t1h].e Cdoensicgonmaintadntdlyis, cmovoreerystoruf cimtuprarlodvaetda, mseacyonbde-ngeendereadtitoonaaidaRthSevdaerisaignnts.and discovery of improved, second-generation aaRS variants
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