Victoria L. Linthwaite's research while affiliated with Durham University and other places

Light harvesting is fundamental for production of ATP and reducing equivalents for CO2 fixation during photosynthesis. However, electronic energy transfer (EET) through a photosystem can harm the photosynthetic apparatus when not balanced with CO2. Here, we show that CO2 binding to the light-harvesting complex modulates EET in photosynthetic cyanobacteria. More specifically, CO2 binding to the allophycocyanin alpha subunit of the light-harvesting complex regulates EET and its fluorescence quantum yield in the cyanobacterium Synechocystis sp. PCC 6803. CO2 binding decreases the inter-chromophore distance in the allophycocyanin trimer. The result is enhanced EET in vitro and in live cells. Our work identifies a direct target for CO2 in the cyanobacterial light-harvesting apparatus and provides insights into photosynthesis regulation. The transfer of electronic energy through a photosystem can harm the photosynthetic apparatus when not balanced with CO2 fixation. Here, the authors show that CO2 modulates electronic energy transfer in cyanobacteria by binding to and enhancing the activity of the light-harvesting complex.

The identification of CO2-binding proteins is crucial to understanding CO2-regulated molecular processes. CO2 can form a reversible posttranslational modification through carbamylation of neutral N-terminal α-amino or lysine ε-amino groups. We have previously developed triethyloxonium (TEO) ion as a chemical proteomics tool for covalent trapping of carbamates, and here, we deploy TEO to identify ubiquitin as a mammalian CO2-binding protein. We use 13C-NMR spectroscopy to demonstrate that CO2 forms carbamates on the ubiquitin N terminus and ε-amino groups of lysines 6, 33, 48, and 63. We demonstrate that biologically relevant pCO2 levels reduce ubiquitin conjugation at lysine-48 and down-regulate ubiquitin-dependent NF-κB pathway activation. Our results show that ubiquitin is a CO2-binding protein and demonstrates carbamylation as a viable mechanism by which mammalian cells can respond to fluctuating pCO2.

A new method for facilitating the delivery, uptake and intracellular localisation of thermally activated delayed fluorescence (TADF) complexes was developed. First, confinement of TADF complexes in liposomes was demonstrated, which were subsequently used as the delivery vehicle for cellular uptake. Confocal fluorescence microscopy showed TADF complexes subsequently localise in the cytoplasm of HepG2 cells. The procedures developed in this work included the removal of molecular oxygen in the liposome preparation without disrupting the liposome structures. Time-resolved fluorescence microscopy (point scanning) showed initial prompt fluorescence followed by a weak, but detectable, delayed fluorescence component for liposomal TADF internalised in HepG2 cells. By demonstrating that it is possible to deliver un-functionalised and/or unshielded TADF complexes, a sensing function for TADFs, such as molecular oxygen, can be envisaged.

Carbon dioxide can influence cell phenotypes through the modulation of signalling pathways. CO 2 regulates cellular processes as diverse as metabolism, cellular homeostasis, chemosensing and pathogenesis. This diversity of regulated processes suggests a broadly conserved mechanism for CO 2 interactions with diverse cellular targets. CO 2 is generally unreactive but can interact with neutral amines on protein under normal intracellular conditions to form a carbamate post-translational modification (PTM). We have previously demonstrated the presence of this PTM in a subset of protein produced by the model plant species Arabidopsis thaliana . Here, we describe a detailed methodology for identifying new carbamate PTMs in an extracted soluble proteome under biologically relevant conditions. We apply this methodology to the soluble proteome of the model prokaryote Escherichia coli and identify new carbamate PTMs . The application of this methodology, therefore, supports the hypothesis that the carbamate PTM is both more widespread in biology than previously suspected and may represent a broadly relevant mechanism for CO 2 –protein interactions.

CO 2 is the inevitable by-product of oxidative metabolism. Many physiological processes such as breathing and cerebral blood flow are sensitive to CO 2 . Historically, the physiological actions of CO 2 have been regarded as being mediated exclusively via changes in pH. Here, we change this consensus by showing that the gap junction protein Connexin26 (Cx26) acts as a receptor for CO 2 showing sensitivity to modest changes in PCO 2 around the physiological norm. Mass spectrometry analysis shows that CO 2 carbamylates specific lysines on a regulatory loop of Cx26 at high, but not at low levels of PCO 2 . By means of high resolution cryo-EM, we have solved structures of Cx26 gap junctions at 1.9, 2.2 and 2.1 Å for PCO 2 of 90, 55 and 20 mmHg respectively, all at pH 7.4. Classification of the particles at each level of PCO 2 , shows the transmembrane helices and N-terminal helix flexing at the dynamic cytoplasmic side of the protein. Gating of Cx26 gap junctions by CO 2 involves movements of the N-terminus to plug the channel at high PCO 2 . We therefore provide mechanistic detail for a new paradigm by which CO 2 can directly control breathing8 and other key physiological functions.

The original version of this Article omitted the following from the Acknowledgements: 'This work was support by EPSRC grant EP/K504336/1 and Leverhulme Trust grant RPG-2016-017.' This has been corrected in both the PDF and HTML versions of the Article.

Carbon dioxide is vital to the chemistry of life processes including metabolism, cellular homoeostasis, and pathogenesis. CO2 is generally unreactive but can combine with neutral amines to form carbamates on proteins under physiological conditions. The most widely known examples of this are CO2 regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase and haemoglobin. However, the systematic identification of CO2-binding sites on proteins formed through carbamylation has not been possible due to the ready reversibility of carbamate formation. Here we demonstrate a methodology to identify protein carbamates using triethyloxonium tetrafluoroborate to covalently trap CO2, allowing for downstream proteomic analysis. This report describes the systematic identification of carbamates in a physiologically relevant environment. We demonstrate the identification of carbamylated proteins and the general principle that CO2 can impact protein biochemistry through carbamate formation. The ability to identify protein carbamates will significantly advance our understanding of cellular CO2 interactions.