Biology subjects

Whelan, F.

Publications and source records attributed to Whelan, F..

5 recordsLinked to original sources

bioRxiv2025

Defining bottlenecks and physiological impact of an orthogonal translation initiation system

Reengineering translation initiation provides a powerful route to develop new translation systems that enable precise control of protein synthesis. While many engineered translation systems show promise, their orthogonality and impact on host physiology is largely uncharacterized, limiting broader application. Here, we develop an initiator-tRNA with an AAC anticodon mutation (i-tRNA-AAC), enabling translation initiation at a GUU start codon. Using fluorescence assays, proteomics, and tRNA sequencing, we assess the i-tRNA-AAC initiation orthogonality and effects on the host, guiding the optimization of its translational efficiency. We find the i-tRNA-AAC mutant initiates translation exclusively from its GUU start codon and is improved by overexpression of valyl-tRNA synthetase and methionyl-tRNA formyltransferase. However, this intervention perturbs aminoacylation and base modifications of endogenous tRNAs along with proteome-wide changes likely due to increased valine demand. Our findings demonstrate how an orthogonal translation initiation system reshapes host physiology and reveals the adaptive responses that accompany translational reprogramming.

synthetic biology↗

bioRxiv2024

The S-lignin O-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

The O-demethylation of lignin aromatics is a rate-limiting step in their bioconversion to high-value compounds. A recently discovered cytochrome P450 enzyme SyoA was found to demethylate the sinapyl alcohol-derived (S-lignin) aromatic syringol. In this work, we solved high-resolution X-ray crystal structures of SyoA in the substrate-free and substrate-bound states and evaluate the demethylation of para-substituted S-lignin aromatics via the monooxygenase pathway and peroxide shunt pathway. We found that SyoA demethylates S-lignin aromatics with the following activity: 4-methylsyringol > syringaldehyde > syringol exclusively using the peroxide shunt pathway. The atomic-resolution structure of SyoA reveals the position of the non-canonical residues in the I-helix (Gln252 and Glu253). Site-directed mutagenesis of this amide-acid pair of a homologous CYP255 enzyme GcoA, which can catalyze the O-demethylation of guaiacol using both monooxygenase and peroxygenase activity, showed the amide-acid pair is critical for both pathways. This work expands the enzymatic toolkit for improving the capacity to funnel lignin towards high-value compounds, and defines the new chemistry within the active site of the enzyme that enables efficient peroxygenase activity. These insights provide a framework for engineered peroxygenase activity in other cytochrome P450 enzymes, with the potential for more facile catalysis, relative to traditional P450 monooxygenases which require difficult to handle redox partners and expensive nicotinamide cofactors.

biochemistry↗

bioRxiv2021

Orthogonal translation initiation using the non-canonical initiator tRNA(AAC) alters protein sequence and stability in vivo

Biological engineers seek to have better control and a more complete understanding of the process of translation initiation within cells so that they may produce proteins more efficiently, as well as to create orthogonal translation systems. Previously, initiator tRNA variants have been created that initiate translation from non-AUG start codons, but their orthogonality has never been measured and the detailed characteristics of proteins produced from them have not been well defined. In this study we created an initiator tRNA mutant with anticodon altered to AAC to be complementary to GUU start codons. We deploy this i-tRNA(AAC) into E. coli cells and measure translation initiation efficiency against all possible start codons. Using parallel reaction monitoring targeted mass spectrometry we identify the N-terminal amino acids of i-tRNA(AAC)-initiated reporter proteins and show these proteins have altered stability within cells. We also use structural modeling of the peptide deformylase enzyme interaction with position 1 valine peptides to interrogate a potential mechanism for accumulation of formylated-valine proteins observed by mass spectrometry. Our results demonstrate that mutant initiator tRNAs have potential to initiate translation more orthogonally than the native initiator tRNA but their interactions with cellular formyltransferases and peptide deformylases can be inefficient because of the amino acid they are charged with. Additionally, engineered initiator tRNAs may enable tuning of in vivo protein stability through initiation with non-methionine amino acids that alter their interaction with cellular proteases.

synthetic biology↗

bioRxiv2020

Periscope Proteins are variable length regulators of bacterial cell surface interactions

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly-available long-read genome sequencing data, we propose this new class could contain over 50 distinct proteins, including those implicated in host colonisation and biofilm formation by human pathogens. In large multi-domain proteins, sequence divergence between adjacent domains appears to reduce inter-domain misfolding. Periscope Proteins break this "rule", suggesting their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria and the immune system.

microbiology↗

bioRxiv2019

Reconstructing and Analysing The Genome of The Last Eukaryote Common Ancestor to Better Understand the Transition from FECA to LECA.

It is still a matter of debate whether the First Eukaryote Common Ancestor (FECA) arose from the merger of an archaeal host with an alphaproteobacterium, or was a proto-eukaryote with significant eukaryotic characteristics way before endosymbiosis occurred. The Last Eukaryote Common Ancestor (LECA) as its descendant is thought to be an entity that possessed functional and cellular complexity comparable to modern organisms. The precise nature and physiology of both of these organisms has been a long-standing, unanswered question in evolutionary and cell biology. Recently, a much broader diversity of eukaryotic genomes has become available and this means we can reconstruct early eukaryote evolution with a greater deal of precision. Here, we reconstruct a hypothetical genome for LECA from modern eukaryote genomes. The constituent genes were mapped onto 454 pathways from the KEGG database covering cellular, genetic, and metabolic processes across six model species to provide functional insights into its capabilities. We reconstruct a LECA that was a facultatively anaerobic, single-celled organism, similar to a modern Protist possessing complex predatory and sexual behaviour. We go on to examine how much of these capabilities arose along the FECA-to-LECA transition period. We see a at least 1,554 genes gained by FECA during this evolutionary period with extensive remodelling of pathways relating to lipid metabolism, cellular processes, genetic information processing, protein processing, and signalling. We extracted the BRITE classifications for the genes from the KEGG database, which arose during the transition from FECA-to-LECA and examine the types of genes that saw the most gains and what novel classifications were introduced. Two-thirds of our reconstructed LECA genome appears to be prokaryote in origin and the remaining third consists of genes with functional classifications that originate from prokaryote homologs in our LECA genome. Signal transduction and Post Translational Modification elements stand out as the primary novel classes of genes developed during this period. These results suggest that largely the eukaryote common ancestors achieved the defining characteristics of modern eukaryotes by primarily expanding on prokaryote biology and gene families.

evolutionary biology↗