A methodology for carbamate post-translational modification discovery and its application in Escherichia coli

Abstract

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.

Carbon dioxide enhances sulphur-selective conjugate addition reactions

“…Although it has been reported that CO 2 -amine interactions on proteins can alter their physical and chemical properties, no attempts have been made to apply this as a synthetic method. 13 a ,25 We first investigated pentapeptide modification with FKVCF (Phe-Lys-Val-Cys-Phe) 26 with 1e under CO 2 and N 2 atmosphere. We detected conjugated product FKVCF- 1e after HPLC purification and by LC/MS analysis.…”

Carbon Dioxide and the Carbamate Post-Translational Modification

“…lipid-transfer protein (Lys-K65), Rubisco Large Subunit (Lys-185), Peroxidase (Lys-262 and Lys-268), FBA1 (Lys-293), eukaryotic aspartyl protease family protein (Lys-251), PSBQA (Lys-109), and Fe Superoxide dismutase 1 (Lys-208) (Linthwaite et al, 2018)). Similarly, a screen of an Escherichia coli lysates corresponding to 14% of the total proteome (294/4300 proteins) identified six CO 2 -binding sites (Linthwaite and Cann, 2021). In this instance, these included proteins involved in cellular processes identified as responsive to CO 2, i.e.…”

Carbon dioxide detection in biological systems

“…While many pathways are implicated as sensitive to changes in CO 2 , a discovery method is required to identify sites of CO 2 interaction within these pathways. Linthwaite & Cann [1] present research describing the use of state-of-the-art technology to discover CO 2 binding proteins within a soluble proteome and apply this methodology to Escherichia coli . This method has been previously deployed using the soluble leaf proteome of Arabidopsis thaliana with similar results: the discovery of a selection of proteins previously unknown to interact with CO 2 directly.…”