Abstract
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.
Highlights
The work of Lorimer and co-workers provided a mechanism for carbamate formation whereby nucleophilic attack of a neutral amine on CO2 converts the amine to an anionic group with the possibility for modulating protein activity[14] (Fig. 1a)
The identification of carbamylation as a PTM in haemoglobin and RuBisCO led to the proposal that carbamylation of neutral Nterminal α-amino groups and the ε-amino group of lysine side chains could form the basis of a widespread mechanism for biological regulation[16]
We describe the general principle that CO2 can reversibly bind protein through carbamate formation
Summary
The work of Lorimer and co-workers provided a mechanism for carbamate formation whereby nucleophilic attack of a neutral amine on CO2 converts the amine to an anionic group with the possibility for modulating protein activity[14] (Fig. 1a). In support of a general biological relevance for such a mechanism of CO2-binding in protein, several carbamates have been identified on lysine side chains in crystal structures including urease[18], alanine racemase[19], transcarboxylase 5 S20, class D β-lactamase[21], and phosphotriesterase[22]. Carbamate formation on Lys[125] is hypothesised to stabilise an electrostatic interaction with Arg[104] in the neighbouring subunit of the hexamer constraining the hemi-channel in the open state in response to high [CO2]. The lability of such exchangeable carbamates outside of the cellular environment makes the identification of this PTM a significant analytical challenge. We demonstrate the general principle that the activity of an identified protein is altered in response to CO2 on mutation of the identified CO2-binding site
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