Biology subjects

Ad, O.

Publications and source records attributed to Ad, O..

6 recordsLinked to original sources

bioRxiv2022

Orthogonal synthetases for polyketide precursors

The absence of orthogonal aminoacyl-tRNA synthetases that accept non-L--amino acids is the primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers. Here we report PylRS enzymes that accept -hydroxy acids, -thio acids, N-formyl-L--amino acids, and -carboxyl acid monomers (malonic acids) that are formally precursors to polyketide natural products. These monomers are all accommodated and accepted by the translation apparatus in vitro. High-resolution structural analysis of the complex between one such PylRS enzyme and a meta-substituted 2-benzylmalonate derivative reveals an active site that discriminates pro-chiral carboxylates and accommodates the large size and distinct electrostatics of an -carboxyl acid substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose -substituent diverges significantly from the -amine embodied in proteinogenic amino acids. These enzymes could act in synergy with natural or evolved ribosomes to generate diverse sequence-defined non-protein hetero-oligomers.

synthetic biology↗

bioRxiv2022

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

biophysics↗

bioRxiv2021

Engineering site-selective incorporation of fluorine into natural product analogs

While bioactive compounds are commonly derived both by human design as well as from living organisms, man-made and natural products typically display very different structural characteristics. As such, a longstanding goal in the discovery of new molecular function is to develop approaches to incorporate the advantageous elements of both groups of molecules, thereby expanding the molecular space accessible for this purpose. In this work, we report the engineering a fluorine-selective enzyme that can complement mutated acyltransferase (AT) domains of a modular polyketide synthase, which are the main determinants of the identity and location of substituents on polyketides, to produce different fluorinated regioisomers of the erythromycin precursor in vitro. We further show that by engineering cell uptake of fluorinated building blocks, we can control fluorine selectivity in vivo to produce selectively fluorinated polyketides using engineered E. coli. These results demonstrate that it is possible to introduce fluorine, a key synthetic design element for drug development, selectively into the scaffold of a complex natural product and produce these analogs by microbial fermentation.

synthetic biology↗

bioRxiv2021

Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

We report that the recently introduced commercial strain of V. natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 non-canonical -amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than10-fold higher than those achieved using two different genomically recoded E. coli strains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.{Delta}A.opt and C321.{Delta}A.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of mis-incorporated natural -amino acids at the UAG-programmed position, especially in cases where the ncAA is an imperfect substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve specific activity of highly desirable but imperfect ncAA substrates.

synthetic biology↗

bioRxiv2020

Structure of the Bacterial Ribosome at 2 A Resolution

Continuing advances in cryo-electron microscopy (cryo-EM) demonstrate the promise it holds for revealing biological structures at chemical resolution, in which noncovalent interactions, RNA and protein modifications, and solvation can be modeled accurately. At present, the best cryo-EM-derived models of the bacterial ribosome are of the large (50S) ribosomal subunit with effective global resolutions of 2.4-2.5 [A], based on map-to-model Fourier shell correlation (FSC). Here we present a model of the E. coli 70S ribosome with an effective global resolution of 2.0 [A], based on maps showcasing unambiguous positioning of residues, their detailed chemical interactions, and chemical modifications. These modifications include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former of which is likely conserved in all domains of life. The model also defines extensive solvation of the small (30S) ribosomal subunit for the first time, as well as interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The high quality of the maps now allows a deeper phylogenetic analysis of ribosomal components, and identification of structural conservation to the level of solvation. The maps and models of the bacterial ribosome presented here should enable future structural analysis of the chemical basis for translation, and the development of robust tools for cryo-EM structure modeling and refinement.

biophysics↗

bioRxiv2019

Defects in the assembly of ribosomes selected for β-amino acid incorporation

Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several non-standard monomers including D-amino acids, dipeptides, and {beta}-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo {beta}-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering towards new catalytic abilities.

biochemistry↗