Publications


  • Crnković, A., Vargas-Rodriguez, O., Merkuryev, A., & Söll, D. (2018). Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids. Bioengineering (Basel, Switzerland), 5(1), 11. https://doi.org/10.3390/bioengineering5010011

    Link

    Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep). The incorporation of Sep into a green fluorescent protein (GFP) in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS) increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.

    @article{crnkovic2018effects,
      author = {Crnkovi{\'{c}}, Ana and Vargas-Rodriguez, Oscar and Merkuryev, Anna and S{\"{o}}ll, Dieter},
      doi = {10.3390/bioengineering5010011},
      issn = {2306-5354},
      journal = {Bioengineering (Basel, Switzerland)},
      keywords = {aminoacyl-tRNA synthetases,genetic code expansion,noncanonical amino acids,phosphoserine,posttranscriptional modifications,protein translation,tRNA},
      number = {1},
      pages = {11},
      pmid = {29393901},
      publisher = {Multidisciplinary Digital Publishing Institute},
      title = {{Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids.}},
      url = {http://www.ncbi.nlm.nih.gov/pubmed/29393901{\%}0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC5874877},
      volume = {5},
      year = {2018}
    }
    
  • Davis López, S. A., Griffith, D. A., Choi, B., Cate, J. H. D., & Tullman-Ercek, D. (2018). Evolutionary engineering improves tolerance for medium-chain alcohols in Saccharomyces cerevisiae. Biotechnology for Biofuels, 11(1). https://doi.org/10.1186/s13068-018-1089-9

    Link

    Yeast-based chemical production is an environmentally friendly alternative to petroleum-based production or processes that involve harsh chemicals. However, many potential alcohol biofuels, such as n-butanol, isobutanol and n-hexanol, are toxic to production organisms, lowering the efficiency and cost-effectiveness of these processes. We set out to improve the tolerance of Saccharomyces cerevisiae toward these alcohols. We evolved the laboratory strain of S. cerevisiae BY4741 to be more tolerant toward n-hexanol and show that the mutations which confer tolerance occur in proteins of the translation initiation complex. We found that n-hexanol inhibits initiation of translation and evolved mutations in the αsubunit of eIF2 and the γsubunit of its guanine exchange factor eIF2B rescue this inhibition. We further demonstrate that translation initiation is affected by other alcohols such as n-pentanol and n-heptanol, and that mutations in the eIF2 and eIF2B complexes greatly improve tolerance to these medium-chain alcohols. We successfully generated S. cerevisiae strains that have improved tolerance toward medium-chain alcohols and have demonstrated that the causative mutations overcome inhibition of translation initiation by these alcohols.

    @article{lopez2018evolutionary,
      author = {{Davis L{\'{o}}pez}, Stephanie A. and Griffith, Douglas Andrew and Choi, Brian and Cate, Jamie H.D. and Tullman-Ercek, Danielle},
      doi = {10.1186/s13068-018-1089-9},
      issn = {17546834},
      journal = {Biotechnology for Biofuels},
      keywords = {Alcohol tolerance,Biofuels,Medium-chain alcohols,Saccharomyces cerevisiae,Translation initiation,eIF2,eIF2B},
      number = {1},
      pmid = {29619086},
      publisher = {BIOMED CENTRAL LTD 236 GRAYS INN RD, FLOOR 6, LONDON WC1X 8HL, ENGLAND},
      title = {{Evolutionary engineering improves tolerance for medium-chain alcohols in Saccharomyces cerevisiae}},
      url = {https://dx.doi.org/10.1186/s13068-018-1089-9},
      volume = {11},
      year = {2018}
    }
    
  • Fu, X., Söll, D., & Sevostyanova, A. (2018). Challenges of site-specific selenocysteine incorporation into proteins by Escherichia coli. RNA Biology, 6286, 01–24. https://doi.org/10.1080/15476286.2018.1440876

    Link

    Selenocysteine (Sec), a rare genetically encoded amino acid with unusual chemical properties, is of great interest for protein engineering. Sec is synthesized on its cognate tRNA (tRNASec) by the concerted action of several enzymes. While all other aminoacyl-tRNAs are delivered to the ribosome by the elongation factor Tu (EF-Tu), Sec-tRNASec requires a dedicated factor, SelB. Incorporation of Sec into protein requires recoding of the stop codon UGA aided by a specific mRNA structure, the SECIS element. This unusual biogenesis restricts the use of Sec in recombinant proteins, limiting our ability to study the properties of selenoproteins. Several methods are currently available for the synthesis selenoproteins. Here we focus on strategies for in vivo Sec insertion at any position(s) within a recombinant protein in a SECIS-independent manner: (i) engineering of tRNASec for use by EF-Tu without the SECIS requirement, and (ii) design of a SECIS-independent SelB route.

    @article{fu2018challenges,
      author = {Fu, Xian and S{\"{o}}ll, Dieter and Sevostyanova, Anastasia},
      doi = {10.1080/15476286.2018.1440876},
      issn = {1547-6286},
      journal = {RNA Biology},
      pages = {01--24},
      publisher = {Taylor {\&} Francis},
      title = {{Challenges of site-specific selenocysteine incorporation into proteins by Escherichia coli}},
      url = {https://www.tandfonline.com/doi/full/10.1080/15476286.2018.1440876},
      volume = {6286},
      year = {2018}
    }
    
  • Glaeser, R. M., Han, B.-G., Watson, Z., Ward, F., & Cate, J. H. D. (2018). Streptavidin Affinity Grids for cryo-EM. Biophysical Journal, 114(3), 163a. https://doi.org/10.1016/j.bpj.2017.11.911

    Link

    The preparation of specimen grids for cryo-EM often proves to be difficult because of unwanted factors such as preferential orientation of particles, too few particles being seen within holes, particle disruption occurring within thin aqueous films, and unexpected aggregation of sample material. In instances where it is suspected that those difficulties might be caused by interaction with the air-water interface, a possible solution is to immobilize particles on a structure-friendly support film, thus preventing diffusion to the air-water interface of the thin aqueous film that remains after blotting away excess sample.

    @article{glaeser2018streptavidin,
      author = {Glaeser, Robert M and Han, Bong-Gyoon and Watson, Zoe and Ward, Fred and Cate, Jamie H.D.},
      doi = {10.1016/j.bpj.2017.11.911},
      issn = {00063495},
      journal = {Biophysical Journal},
      month = feb,
      number = {3},
      pages = {163a},
      publisher = {Elsevier},
      title = {{Streptavidin Affinity Grids for cryo-EM}},
      url = {http://linkinghub.elsevier.com/retrieve/pii/S0006349517321434},
      volume = {114},
      year = {2018}
    }
    
  • Ho, J. M., Bakkalbasi, E., Söll, D., & Miller, C. A. (2018). Drugging tRNA aminoacylation. RNA Biology, 1–11. https://doi.org/10.1080/15476286.2018.1429879

    Link

    Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.

    @article{ho2018drugging,
      author = {Ho, Joanne M. and Bakkalbasi, Erol and S{\"{o}}ll, Dieter and Miller, Corwin A.},
      doi = {10.1080/15476286.2018.1429879},
      issn = {1547-6286},
      journal = {RNA Biology},
      keywords = {Aminoacylation,aminoacyl tRNA synthetases,antibiotic targets,antibiotics,antimicrobials,drug development,drug targets,transfer RNA,translation inhibitors},
      month = feb,
      pages = {1--11},
      pmid = {29345185},
      publisher = {Taylor {\&} Francis},
      title = {{Drugging tRNA aminoacylation}},
      url = {https://www.tandfonline.com/doi/full/10.1080/15476286.2018.1429879},
      year = {2018}
    }
    
  • Kim, H., Oh, E. J., Lane, S. T., Lee, W.-H., Cate, J. H. D., & Jin, Y.-S. (2018). Enhanced cellobiose fermentation by engineered Saccharomyces cerevisiae expressing a mutant cellodextrin facilitator and cellobiose phosphorylase. Journal of Biotechnology, 275, 53–59. https://doi.org/10.1016/j.jbiotec.2018.04.008

    Link

    To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast.

    @article{kim2018enhanced,
      author = {Kim, Heejin and Oh, Eun Joong and Lane, Stephan Thomas and Lee, Won-Heong and Cate, Jamie H.D. and Jin, Yong-Su},
      doi = {10.1016/j.jbiotec.2018.04.008},
      issn = {01681656},
      journal = {Journal of Biotechnology},
      keywords = {Cellobiose fermentation,Cellobiose phosphorylase,Cellodextrin transporter,Cerevisiae},
      month = jun,
      pages = {53--59},
      publisher = {Elsevier},
      title = {{Enhanced cellobiose fermentation by engineered Saccharomyces cerevisiae expressing a mutant cellodextrin facilitator and cellobiose phosphorylase}},
      url = {http://linkinghub.elsevier.com/retrieve/pii/S0168165618301184},
      volume = {275},
      year = {2018}
    }
    
  • Londregan, A. T., Wei, L., Xiao, J., Lintner, N. G., Petersen, D., Dullea, R. G., … Piotrowski, D. W. (2018). Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents. Journal of Medicinal Chemistry. https://doi.org/10.1021/acs.jmedchem.8b00650

    Link

    The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.

    @article{londregan2018small,
      author = {Londregan, Allyn Timothy and Wei, Liuqing and Xiao, Jun and Lintner, Nathanael G and Petersen, Donna and Dullea, Robert G. and McClure, Kim F. and Bolt, Michael W and Warmus, Joseph S and Coffey, Steven B and Limberakis, Chris and Genovino, Julien and Thuma, Benjamin A and Hesp, Kevin D and Aspnes, Gary E and Reidich, Benjamin and Salatto, Christopher T and Chabot, Jeffrey R and Cate, Jamie H D and Liras, Spiros and Piotrowski, David W},
      doi = {10.1021/acs.jmedchem.8b00650},
      issn = {1520-4804},
      journal = {Journal of medicinal chemistry},
      month = jun,
      pmid = {29878763},
      publisher = {ACS Publications},
      title = {{Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents.}},
      url = {http://www.ncbi.nlm.nih.gov/pubmed/29878763},
      year = {2018}
    }
    
  • Melnikov, S. V., Rivera, K. D., Ostapenko, D., Makarenko, A., Sanscrainte, N. D., Becnel, J. J., … Söll, D. (2018). Error-prone protein synthesis in parasites with the smallest eukaryotic genome. Proceedings of the National Academy of Sciences, 115(27), E6245–E6253. https://doi.org/10.1073/pnas.1803208115

    Link

    Microsporidia are parasitic fungi-like organisms that invade the interior of living cells and cause chronic disorders in a broad range of animals, including humans. These pathogens have the tiniest known genomes among eukaryotic species, for which they serve as a model for exploring the phenomenon of genome reduction in obligate intracellular parasites. Here we report a case study to show an apparent effect of overall genome reduction on the primary structure and activity of aminoacyl-tRNA synthetases, indispensable cellular proteins required for protein synthesis. We find that most microsporidian synthetases lack regulatory and eukaryote-specific appended domains and have a high degree of sequence variability in tRNA-binding and catalytic domains. In one synthetase, LeuRS, an apparent sequence degeneration annihilates the editing domain, a catalytic center responsible for the accurate selection of leucine for protein synthesis. Unlike accurate LeuRS synthetases from other eukaryotic species, microsporidian LeuRS is error-prone: apart from leucine, it occasionally uses its near-cognate substrates, such as norvaline, isoleucine, valine, and methionine. Mass spectrometry analysis of the microsporidium Vavraia culicis proteome reveals that nearly 6% of leucine residues are erroneously replaced by other amino acids. This remarkably high frequency of mistranslation is not limited to leucine codons and appears to be a general property of protein synthesis in microsporidian parasites. Taken together, our findings reveal that the microsporidian protein synthesis machinery is editing-deficient, and that the proteome of microsporidian parasites is more diverse than would be anticipated based on their genome sequences.

    @article{melnikov2018error,
      author = {Melnikov, Sergey V and Rivera, Keith D and Ostapenko, Denis and Makarenko, Arthur and Sanscrainte, Neil D and Becnel, James J and Solomon, Mark J and Texier, Catherine and Pappin, Darryl J and S{\"{o}}ll, Dieter},
      doi = {10.1073/pnas.1803208115},
      issn = {0027-8424},
      journal = {Proceedings of the National Academy of Sciences},
      month = jul,
      number = {27},
      pages = {E6245--E6253},
      publisher = {National Acad Sciences},
      title = {{Error-prone protein synthesis in parasites with the smallest eukaryotic genome}},
      url = {http://www.pnas.org/lookup/doi/10.1073/pnas.1803208115},
      volume = {115},
      year = {2018}
    }
    
  • Melnikov, S., Manakongtreecheep, K., & Söll, D. (2018). Revising the structural diversity of ribosomal proteins across the three domains of life. Molecular Biology and Evolution, 35(March), 1–27. https://doi.org/10.1093/MOLBEV/MSY021

    Link

    Ribosomal proteins are indispensable components of a living cell, and yet their structures are remarkably diverse in different species. Here we use manually curated structural alignments to provide a comprehensive catalog of structural variations in homologous ribosomal proteins from bacteria, archaea, eukaryotes and eukaryotic organelles. By resolving numerous ambiguities and errors of automated structural and sequence alignments, we uncover a whole new class of structural variations, which reside within seemingly conserved segments of ribosomal proteins. We then illustrate that these variations reflect an apparent adaptation of ribosomal proteins to the specific environments and lifestyles of living species. Finally, we show that most of these structural variations reside within non-globular extensions of ribosomal proteins {\backslashtextendash} protein segments that are thought to promote ribosome biogenesis by stabilizing the proper folding of ribosomal RNA. We show that, even though the extensions are thought to be the most ancient peptides on our planet, they are in fact the most rapidly evolving and most structurally and functionally diverse segments of ribosomal proteins. Overall, our work illustrates that, despite being long considered as slowly evolving and highly conserved, ribosomal proteins are more complex and more specialized than is generally recognized.

    @article{melnikov2018revising,
      author = {Melnikov, Sergey and Manakongtreecheep, Kasidet and S{\"{o}}ll, Dieter},
      doi = {10.1093/MOLBEV/MSY021},
      issn = {0737-4038},
      journal = {Molecular Biology and Evolution},
      number = {March},
      pages = {1--27},
      pmid = {29529322},
      publisher = {Oxford University Press},
      title = {{Revising the structural diversity of ribosomal proteins across the three domains of life}},
      url = {https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msy021/4908658},
      volume = {35},
      year = {2018}
    }
    
  • Mukai, T., Sevostyanova, A., Suzuki, T., Fu, X., & Söll, D. (2018). Eine einfache Methode zur Produktion von Selenoproteinen. Angewandte Chemie, 130(24), 7333–7337. https://doi.org/10.1002/ange.201713215

    Link

    Ein einfacher Ansatz nutzt einen erweiterten genetischen Code von Escherichia coli zur Biosynthese von Selenoproteinen mit zahlreichen Sec‐Resten. Kürzlich wurden so genannte allo‐tRNAs entdeckt. Diese verfügen über eine ungewöhnliche Struktur, sind genauso effiziente Serinakzeptoren wie die normale tRNASer aus E. coli und werden von der Aeromonas‐salmonicida‐Selenocysteinsynthase (SelA) von Ser‐allo‐tRNA zu Sec‐allo‐tRNA umgesetzt. Anschließend ermöglicht es Sec‐allo‐tRNA, fünf UAG‐Stop‐Codons auf der fdhF‐mRNA für E.‐coli‐Formatdehydrogenase H als Sec zu translatieren und katalytisch aktive E.‐coli‐Formatdehydrogenase mit fünf Sec‐Resten in E. coli zu produzieren. Weiterhin konnte gezeigt werden, dass sich in E. coli durch Kombination genetischer Varianten von allo‐tRNA und SelA mit einem modifizierten Selenstoffwechsel das humane Selenoenzym GPx1 mit über 80 % Sec‐Einbaurate rekombinant produzieren lässt. Beide Beispiele belegen den Wert von allo‐tRNAUTu als molekulare Plattform zur Entwicklung neuartiger Selenoproteine.

    @article{mukai2018einfache,
      author = {Mukai, Takahito and Sevostyanova, Anastasia and Suzuki, Tateki and Fu, Xian and S{\"{o}}ll, Dieter},
      doi = {10.1002/ange.201713215},
      issn = {00448249},
      journal = {Angewandte Chemie},
      month = jun,
      number = {24},
      pages = {7333--7337},
      publisher = {Wiley Online Library},
      title = {{Eine einfache Methode zur Produktion von Selenoproteinen}},
      url = {http://doi.wiley.com/10.1002/ange.201713215},
      volume = {130},
      year = {2018}
    }
    
  • Mukai, T., Sevostyanova, A., Suzuki, T., Fu, X., & Söll, D. (2018). A Facile Method for Producing Selenocysteine-Containing Proteins. Angewandte Chemie International Edition, 57(24), 7215–7219. https://doi.org/10.1002/anie.201713215

    Link

    Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNASer. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDHH with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNAUTu system offers a new selenoprotein engineering platform.

    @article{mukai2018facile,
      author = {Mukai, Takahito and Sevostyanova, Anastasia and Suzuki, Tateki and Fu, Xian and S{\"{o}}ll, Dieter},
      doi = {10.1002/anie.201713215},
      issn = {14337851},
      journal = {Angewandte Chemie International Edition},
      month = jun,
      number = {24},
      pages = {7215--7219},
      publisher = {Wiley Online Library},
      title = {{A Facile Method for Producing Selenocysteine-Containing Proteins}},
      url = {http://doi.wiley.com/10.1002/anie.201713215},
      volume = {57},
      year = {2018}
    }
    
  • Quach, K., LaRochelle, J., Li, X.-H., Rhoades, E., & Schepartz, A. (2018). Unique arginine array improves cytosolic localization of hydrocarbon-stapled peptides. Bioorganic & Medicinal Chemistry, 26(6), 1197–1202. https://doi.org/10.1016/j.bmc.2017.11.008

    Link

    We have previously reported that miniature proteins containing a distinct array of 5 arginine residues on a folded α-helix – a penta-arg motif – traffic with high efficiency from endosomes into the cytosol and nucleus of mammalian cells. Here we evaluate whether a penta-arg motif can improve the intracellular trafficking of an otherwise impermeant hydrocarbon-stapled peptide, SAH-p53-4Rho. We prepared a panel of SAH-p53-4Rhovariants containing penta-arg sequences with different spacings and axial arrangement and evaluated their overall uptake (as judged by flow cytometry) and their intracellular access (as determined by fluorescence correlation spectroscopy, FCS). One member of this panel reached the cytosol extremely well, matching the level achieved by SAH-p53-8Rho, a previously reported and highly permeant hydrocarbon-stapled peptide. Notably, we found no relationship between cellular uptake as judged by flow cytometry and cytosolic access as determined by FCS. This result reiterates that overall uptake and endosomal release represent fundamentally different biological processes. To determine cytosolic and/or nuclear access, one must measure concentration directly using a quantitative and non-amplified tool such as FCS. As has been observed for highly cell permeant miniature proteins such as ZF5.3, optimal penetration of hydrocarbon-stapled peptides into the cell cytosol results when the penta-arg motif is located within more (as opposed to less) structured regions.

    @article{quach2018unique,
      author = {Quach, Kim and LaRochelle, Jonathan and Li, Xiao-Han and Rhoades, Elizabeth and Schepartz, Alanna},
      doi = {10.1016/j.bmc.2017.11.008},
      issn = {09680896},
      journal = {Bioorganic {\&} Medicinal Chemistry},
      keywords = {Cell-penetrating peptides,Cellular uptake,Fluorescence correlation spectroscopy,Stapled peptide,$\alpha$-Helicity},
      month = mar,
      number = {6},
      pages = {1197--1202},
      publisher = {Pergamon},
      title = {{Unique arginine array improves cytosolic localization of hydrocarbon-stapled peptides}},
      url = {http://linkinghub.elsevier.com/retrieve/pii/S0968089617316334},
      volume = {26},
      year = {2018}
    }
    
  • Schepartz, A. (2018). Foldamers wave to the ribosome. Nature Chemistry, 10(4), 377–379. https://doi.org/10.1038/s41557-018-0036-5

    Link

    Ribosomes have now been shown to accept certain initiator tRNAs acylated with aromatic foldamer–dipeptides thereby enabling the translation of a peptide or protein with a short aromatic foldamer at the N-terminus. Some foldamer–peptide hybrids could be cyclized to generate macrocycles that present conformationally restricted peptide loops.

    @article{schepartz2018foldamers,
      author = {Schepartz, Alanna},
      doi = {10.1038/s41557-018-0036-5},
      issn = {1755-4330},
      journal = {Nature Chemistry},
      month = apr,
      number = {4},
      pages = {377--379},
      title = {{Foldamers wave to the ribosome}},
      url = {http://www.nature.com/articles/s41557-018-0036-5},
      volume = {10},
      year = {2018}
    }
    
  • Sinclair, J. K. L., Walker, A. S., Doerner, A. E., & Schepartz, A. (2018). Mechanism of Allosteric Coupling into and through the Plasma Membrane by EGFR. Cell Chemical Biology. https://doi.org/10.1016/j.chembiol.2018.04.005

    Link

    Epidermal growth factor receptor (EGFR) interacts through its extracellular domain with seven different growth factors. These factors induce different structures within the cytoplasmic juxtamembrane (JM) segment of the dimeric receptor and propagate different growth factor-dependent signals to the cell interior. How this process occurs is unknown. Here we apply diverse experimental and computational tools to show that growth factor identity is encoded by the EGFR transmembrane (TM) helix into discrete helix dimer populations that differ in both cross-location and cross-angle. Helix dimers with smaller cross-angles at multiple cross locations are decoded to induce an EGF-type coiled coil in the adjacent JM, whereas helix dimers with larger cross-angles at fewer cross locations induce the TGF-α-type coiled coil. We propose an updated model for how conformational coupling across multiple EGFR domains results in growth factor-specific information transfer, and demonstrate that this model applies to both EGFR and the related receptor ErbB2. The mechanism by which EGFR communicates growth factor-dependent signals to the cell interior is unknown. Here we show that growth factor identity is encoded into discrete TM helix dimers. These dimers induce different coiled-coil structures within the JM region that correlate with downstream signaling.

    @article{sinclair2018mechanism,
      author = {Sinclair, Julie K.L. and Walker, Allison S. and Doerner, Amy E. and Schepartz, Alanna},
      doi = {10.1016/j.chembiol.2018.04.005},
      issn = {24519456},
      journal = {Cell Chemical Biology},
      keywords = {allostery,biased agonism,cancer,epidermal growth factor receptor (EGFR),information transfer,juxtamembrane domain,ligand-induced dimerization,receptor tyrosine kinase (RTK),signal transduction,transmembrane domain},
      month = may,
      publisher = {Elsevier},
      title = {{Mechanism of Allosteric Coupling into and through the Plasma Membrane by EGFR}},
      url = {http://linkinghub.elsevier.com/retrieve/pii/S2451945618301193},
      year = {2018}
    }
    
  • Vargas-Rodriguez, O., Englert, M., Merkuryev, A., Mukai, T., & Söll, D. (2018). Recoding of the selenocysteine UGA codon by cysteine in the presence of a non-canonical tRNA Cys and elongation factor SelB. RNA Biology, (just-accepted), 1–9. https://doi.org/10.1080/15476286.2018.1474074

    Link

    ABSTRACTIn many organisms, the UGA stop codon is recoded to insert selenocysteine (Sec) into proteins. Sec incorporation in bacteria is directed by an mRNA element, known as the Sec-insertion sequence (SECIS), located downstream of the Sec codon. Unlike other aminoacyl-tRNAs, Sec-tRNASec is delivered to the ribosome by a dedicated elongation factor, SelB. We recently identified a series of tRNASec-like tRNA genes distributed across Bacteria that also encode a canonical tRNASec. These tRNAs contain sequence elements generally recognized by cysteinyl-tRNA synthetase (CysRS). While some of these tRNAs contain a UCA Sec anticodon, most have a GCA Cys anticodon. tRNASec with GCA anticodons are known to recode UGA codons. Here we investigate the clostridial Desulfotomaculum nigrificans tRNASec-like tRNACys, and show that this tRNA is acylated by CysRS, recognized by SelB, and capable of UGA recoding with Cys in Escherichia coli. We named this non-canonical group of tRNACys as ‘tRNAReC’ (Recoding with Cys). We p...

    @article{vargas2018recoding,
      author = {Vargas-Rodriguez, Oscar and Englert, Markus and Merkuryev, Anna and Mukai, Takahito and S{\"{o}}ll, Dieter},
      doi = {10.1080/15476286.2018.1474074},
      issn = {1547-6286},
      journal = {RNA Biology},
      keywords = {Genetic code,bioinformatics,recoding,selenocysteine,tRNA},
      month = jun,
      number = {just-accepted},
      pages = {1--9},
      publisher = {Taylor {\&} Francis},
      title = {{Recoding of the selenocysteine UGA codon by cysteine in the presence of a non-canonical tRNA Cys and elongation factor SelB}},
      url = {https://www.tandfonline.com/doi/full/10.1080/15476286.2018.1474074},
      year = {2018}
    }
    
  • Wadzinski, T. J., Steinauer, A., Hie, L., Pelletier, G., Schepartz, A., & Miller, S. J. (2018). Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent. Nature Chemistry, 10(6), 644–652. https://doi.org/10.1038/s41557-018-0041-8

    Link

    Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

    @article{wadzinski2018rapid,
      author = {Wadzinski, Tyler J. and Steinauer, Angela and Hie, Liana and Pelletier, Guillaume and Schepartz, Alanna and Miller, Scott J.},
      doi = {10.1038/s41557-018-0041-8},
      issn = {1755-4330},
      journal = {Nature Chemistry},
      month = jun,
      number = {6},
      pages = {644--652},
      title = {{Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent}},
      url = {http://www.nature.com/articles/s41557-018-0041-8},
      volume = {10},
      year = {2018}
    }
    
  • Bryson, D. I., Fan, C., Guo, L.-T., Miller, C., Söll, D., & Liu, D. R. (2017). Continuous directed evolution of aminoacyl-tRNA synthetases. Nature Chemical Biology, 13(12), 1253–1260. https://doi.org/10.1038/nchembio.2474

    Link

    Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing noncanonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.

    @article{bryson2017continuous,
      author = {Bryson, David I and Fan, Chenguang and Guo, Li-Tao and Miller, Corwin and S{\"{o}}ll, Dieter and Liu, David R},
      doi = {10.1038/nchembio.2474},
      issn = {1552-4450},
      journal = {Nature Chemical Biology},
      month = oct,
      number = {12},
      pages = {1253--1260},
      publisher = {Nature Publishing Group},
      title = {{Continuous directed evolution of aminoacyl-tRNA synthetases}},
      url = {http://www.nature.com/doifinder/10.1038/nchembio0218-186},
      volume = {13},
      year = {2017}
    }
    
  • Chomvong, K., Benjamin, D. I., Nomura, D. K., & Cate, J. H. D. (2017, September). Cellobiose Consumption Uncouples Extracellular Glucose Sensing and Glucose Metabolism in Saccharomyces cerevisiae. (J. Zhou, Ed.), mBio. https://doi.org/10.1128/mBio.00855-17

    Link

    Glycolysis is central to energy metabolism in most organisms and is highly regulated to enable optimal growth. In the yeast Saccharomyces cerevisiae , feedback mechanisms that control flux through glycolysis span transcriptional control to metabolite levels in the cell. Using a cellobiose consumption pathway, we decoupled glucose sensing from carbon utilization, revealing new modular layers of control that induce ATP consumption to drive rapid carbon fermentation. Alterations of the beta subunit of phosphofructokinase-1 ( PFK2 ), H + -plasma membrane ATPase ( PMA1 ), and glucose sensors ( SNF3 and RGT2 ) revealed the importance of coupling extracellular glucose sensing to manage ATP levels in the cell. Controlling the upper bound of cellular ATP levels may be a general mechanism used to regulate energy levels in cells, via a regulatory network that can be uncoupled from ATP concentrations under perceived starvation conditions.

    @misc{chomvong2017cellobiose,
      author = {Chomvong, Kulika and Benjamin, Daniel I. and Nomura, Daniel K. and Cate, Jamie H. D.},
      booktitle = {mBio},
      doi = {10.1128/mBio.00855-17},
      editor = {Zhou, Jizhong},
      issn = {2150-7511},
      keywords = {Cellobiose,Glucose sensors,Metabolomics,PMA1},
      month = sep,
      number = {4},
      pages = {e00855--17},
      pmid = {28790206},
      title = {{Cellobiose Consumption Uncouples Extracellular Glucose Sensing and Glucose Metabolism in Saccharomyces cerevisiae}},
      url = {http://mbio.asm.org/lookup/doi/10.1128/mBio.00855-17},
      volume = {8},
      year = {2017}
    }
    
  • Erdmann, R. S., Toomre, D., & Schepartz, A. (2017). STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells. In Methods in Molecular Biology (Vol. 1663, pp. 65–78). Springer. https://doi.org/10.1007/978-1-4939-7265-4_6

    Link

    Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are \textgreater100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.

    @incollection{erdmann2017sted,
      author = {Erdmann, Roman S. and Toomre, Derek and Schepartz, Alanna},
      booktitle = {Methods in Molecular Biology},
      doi = {10.1007/978-1-4939-7265-4_6},
      isbn = {978-1-4939-7264-7},
      issn = {10643745},
      keywords = {Bioorthogonal chemistry,Click chemistry,Fluorophores,Inverse electron demand Diels-Alder reaction,Membranes,Super-resolution microscopy},
      pages = {65--78},
      pmid = {28924659},
      publisher = {Springer},
      title = {{STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells}},
      url = {http://link.springer.com/10.1007/978-1-4939-7265-4{\_}6},
      volume = {1663},
      year = {2017}
    }
    
  • Jin, Y.-S., & Cate, J. H. D. (2017). Metabolic engineering of yeast for lignocellulosic biofuel production. Current Opinion in Chemical Biology, 41, 99–106. https://doi.org/10.1016/j.cbpa.2017.10.025

    Link

    Production of biofuels from lignocellulosic biomass remains an unsolved challenge in industrial biotechnology. Efforts to use yeast for conversion face the question of which host organism to use, counterbalancing the ease of genetic manipulation with the promise of robust industrial phenotypes. Saccharomyces cerevisiae remains the premier host for metabolic engineering of biofuel pathways, due to its many genetic, systems and synthetic biology tools. Numerous engineering strategies for expanding substrate ranges and diversifying products of S. cerevisiae have been developed. Other yeasts generally lack these tools, yet harbor superior phenotypes that could be exploited in the harsh processes required for lignocellulosic biofuel production. These include thermotolerance, resistance to toxic compounds generated during plant biomass deconstruction, and wider carbon consumption capabilities. Although promising, these yeasts have yet to be widely exploited. By contrast, oleaginous yeasts such as Yarrowia lipolytica capable of producing high titers of lipids are rapidly advancing in terms of the tools available for their metabolic manipulation.

    @article{jin2017metabolic,
      author = {Jin, Yong-Su and Cate, Jamie HD},
      doi = {10.1016/j.cbpa.2017.10.025},
      isbn = {1367-5931},
      issn = {13675931},
      journal = {Current Opinion in Chemical Biology},
      month = dec,
      pages = {99--106},
      pmid = {29127883},
      publisher = {Elsevier},
      title = {{Metabolic engineering of yeast for lignocellulosic biofuel production}},
      url = {https://linkinghub.elsevier.com/retrieve/pii/S136759311730128X},
      volume = {41},
      year = {2017}
    }
    
  • Lintner, N. G., McClure, K. F., Petersen, D., Londregan, A. T., Piotrowski, D. W., Wei, L., … Cate, J. H. D. (2017). Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain. PLOS Biology, 15(3), e2001882. https://doi.org/10.1371/journal.pbio.2001882

    Link

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.

    @article{lintner2018correction,
      author = {Lintner, Nathanael G. and McClure, Kim F. and Petersen, Donna and Londregan, Allyn T. and Piotrowski, David W. and Wei, Liuqing and Xiao, Jun and Bolt, Michael and Loria, Paula M. and Maguire, Bruce and Geoghegan, Kieran F. and Huang, Austin and Rolph, Tim and Liras, Spiros and Doudna, Jennifer A. and Dullea, Robert G. and Cate, Jamie H. D.},
      doi = {10.1371/journal.pbio.2001882},
      editor = {Khosla, Chaitan},
      isbn = {1111111111},
      issn = {1545-7885},
      journal = {PLOS Biology},
      month = mar,
      number = {3},
      pages = {e2001882},
      pmid = {28323820},
      publisher = {Public Library of Science},
      title = {{Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain}},
      url = {http://dx.plos.org/10.1371/journal.pbio.2001882},
      volume = {15},
      year = {2017}
    }
    
  • Mukai, T., Lajoie, M. J., Englert, M., & Söll, D. (2017). Rewriting the Genetic Code. Annual Review of Microbiology, 71(1), 557–577. https://doi.org/10.1146/annurev-micro-090816-093247

    Link

    The genetic code—the language used by cells to translate their genomes into proteins that perform many cellular functions—is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

    @article{Mukai2017,
      author = {Mukai, Takahito and Lajoie, Marc J. and Englert, Markus and S{\"{o}}ll, Dieter},
      doi = {10.1146/annurev-micro-090816-093247},
      isbn = {1054901536631},
      issn = {0066-4227},
      journal = {Annual Review of Microbiology},
      keywords = {codon usage,genetic code,orthogonal,synthetic biology,translation engineering},
      month = sep,
      number = {1},
      pages = {557--577},
      pmid = {28697669},
      title = {{Rewriting the Genetic Code}},
      url = {http://www.annualreviews.org/doi/10.1146/annurev-micro-090816-093247},
      volume = {71},
      year = {2017}
    }
    
  • Suzuki, T., Miller, C., Guo, L.-T., Ho, J. M. L., Bryson, D. I., Wang, Y.-S., … Söll, D. (2017). Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. Nature Chemical Biology, 13(12), 1261–1266. https://doi.org/10.1038/nchembio.2497

    Link

    The N-terminal domain structure of pyrrolysyl-tRNA synthetase (PylRS) reveals details of its tRNA specificity and facilitates the improvement of its selectivity for non-canonical amino acids by phage-assisted non-continuous evolution (PANCE).

    @article{suzuki2017crystal,
      archiveprefix = {arXiv},
      arxivid = {15334406},
      author = {Suzuki, Tateki and Miller, Corwin and Guo, Li-Tao and Ho, Joanne M L and Bryson, David I. and Wang, Yane-Shih and Liu, David R. and S{\"{o}}ll, Dieter},
      doi = {10.1038/nchembio.2497},
      eprint = {15334406},
      isbn = {1530752558},
      issn = {1552-4450},
      journal = {Nature Chemical Biology},
      month = oct,
      number = {12},
      pages = {1261--1266},
      pmid = {29035363},
      publisher = {Nature Publishing Group},
      title = {{Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase}},
      url = {http://www.nature.com/doifinder/10.1038/nchembio.2497},
      volume = {13},
      year = {2017}
    }
    
  • Thompson, A. D., Bewersdorf, J., Toomre, D., & Schepartz, A. (2017). HIDE Probes: A New Toolkit for Visualizing Organelle Dynamics, Longer and at Super-Resolution. Biochemistry, 56(39), 5194–5201. https://doi.org/10.1021/acs.biochem.7b00545

    Link

    Living cells are complex and dynamic assemblies that carefully sequester and orchestrate multiple diverse processes that enable growth, division, regulation, movement, and communication. Membrane-bound organelles such as the endoplasmic reticulum, mitochondria, plasma membrane, and others are integral to these processes, and their functions demand dynamic reorganization in both space and time. Visualizing these dynamics in live cells over long time periods demands probes that label discrete organelles specifically, at high density, and withstand long-term irradiation. Here we describe the evolution of our work on the development of a set of high-density environmentally sensitive (HIDE) membrane probes that enable long-term, live-cell nanoscopy of the dynamics of multiple organelles in live cells using single-molecule switching and stimulated emission depletion imaging modalities

    @article{thompson2017hide,
      author = {Thompson, Alexander D. and Bewersdorf, Joerg and Toomre, Derek and Schepartz, Alanna},
      doi = {10.1021/acs.biochem.7b00545},
      isbn = {1520-4995 (Electronic) 0006-2960 (Linking)},
      issn = {0006-2960},
      journal = {Biochemistry},
      month = oct,
      number = {39},
      pages = {5194--5201},
      pmid = {28792749},
      publisher = {ACS Publications},
      title = {{HIDE Probes: A New Toolkit for Visualizing Organelle Dynamics, Longer and at Super-Resolution}},
      url = {http://pubs.acs.org/doi/10.1021/acs.biochem.7b00545},
      volume = {56},
      year = {2017}
    }
    
  • Zhang, C., Acosta-Sampson, L., Yu, V. Y., & Cate, J. H. D. (2017). Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae. PLOS ONE, 12(9), e0184730. https://doi.org/10.1371/journal.pone.0184730

    Link

    The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

    @article{zhang2017screening,
      author = {Zhang, Chenlu and Acosta-Sampson, Ligia and Yu, Vivian Yaci and Cate, Jamie H. D.},
      doi = {10.1371/journal.pone.0184730},
      editor = {Yang, Shihui},
      isbn = {1111111111},
      issn = {1932-6203},
      journal = {PLOS ONE},
      month = sep,
      number = {9},
      pages = {e0184730},
      pmid = {28886200},
      publisher = {Public Library of Science},
      title = {{Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae}},
      url = {http://dx.plos.org/10.1371/journal.pone.0184730},
      volume = {12},
      year = {2017}
    }
    
  • Melo Czekster, C., Robertson, W. E., Walker, A. S., Söll, D., & Schepartz, A. (2016). In Vivo Biosynthesis of a β-Amino Acid-Containing Protein. Journal of the American Chemical Society, 138(16), 5194–5197. https://doi.org/10.1021/jacs.6b01023

    Link

    It has recently been reported that ribosomes from erythromycin-resistant Escherichia coli strains, when isolated in S30 extracts and incubated with chemically mis-acylated tRNA, can incorporate certain β-amino acids into full length DHFR in vitro. Here we report that wild-type E. coli EF-Tu and phenylalanyl-tRNA synthetase collaborate with these mutant ribosomes and others to incorporate β(3)-Phe analogs into full length DHFR in vivo. E. coli harboring the most active mutant ribosomes are robust, with a doubling time only 14% longer than wild-type. These results reveal the unexpected tolerance of E. coli and its translation machinery to the β(3)-amino acid backbone and should embolden in vivo selections for orthogonal translational machinery components that incorporate diverse β-amino acids into proteins and peptides. E. coli harboring mutant ribosomes may possess the capacity to incorporate many non-natural, non-α-amino acids into proteins and other sequence-programmed polymeric materials.

    @article{MeloCzekster2016,
      author = {{Melo Czekster}, Clarissa and Robertson, Wesley E. and Walker, Allison S. and S{\"{o}}ll, Dieter and Schepartz, Alanna},
      doi = {10.1021/jacs.6b01023},
      isbn = {0002-7863},
      issn = {15205126},
      journal = {Journal of the American Chemical Society},
      month = apr,
      number = {16},
      pages = {5194--5197},
      pmid = {27086674},
      title = {{In Vivo Biosynthesis of a $\beta$-Amino Acid-Containing Protein}},
      url = {http://pubs.acs.org/doi/abs/10.1021/jacs.6b01023},
      volume = {138},
      year = {2016}
    }