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Development of a method for the investigation of protein carbamylation by CO2

JANUS, JOANNA,MARY (2016) Development of a method for the investigation of protein carbamylation by CO2. Masters thesis, Durham University.

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Protein carbamylation occurs in biological systems when the nitrogen atom of a neutral amine group makes a nucleophilic attack upon the carbon atom of a CO2 molecule. This results in the CO2 becoming bonded to the amine group as a carbamate. Carbamates play important structural and functional roles in several proteins, including haemoglobin, Rubisco and β-lactamases. Despite this and the ubiquity of CO2, relatively few proteins have been shown to be carbamylated. This is partly due to the labile nature of the carbamate bond, meaning it dissociates readily and can be hard to investigate.
A method was developed to ethylate carbamate modifications on proteins using the reagent triethyloxonium tetrafluoroborate (TEO). The ethylation trapped the carbamate modification so it could be analysed by mass spectrometry. It was first confirmed that TEO could trap a carbamate on an amino acid, then the trapping method was adapted for use on proteins. It was shown that the TEO trapping method was able to stabilise a carbamate on haemoglobin which could be detected using MALDI-TOF and electrospray mass spectrometry. The method was also used to test proteins with unknown carbamate forming abilities. Recombinant proteins comprising the catalytic domains of mammalian transmembrane adenylyl cyclases and the related Rv1625c adenylyl cyclase from Mycobacterium tuberculosis were expressed and used in the carbamate trapping experiments. The Rv1625c204-443 protein was revealed to potentially possess a carbamylated Lys-296 residue. Due to inconclusive results an alternative method was used to further explore this result. 14CO2 was supplied to the wild type Rv1625c204-443 recombinant protein and an Rv1625c204-443 protein with Lys-296 mutated to Ala, before potential carbamates were trapped with TEO. It was concluded that it was likely that the Lys-296 residue of Rv1625c204-443 did not form a carbamate, although further work is needed to confirm this result.
A method has therefore been developed to trap carbamate modifications on proteins and has been used to explore whether adenylyl cyclases undergo carbamylation by CO2. The method requires further optimisation and some results were inconclusive. However the findings from this study will contribute towards the development of a method to screen many proteins in a cell lysate for carbamate modifications simultaneously, using proteomic techniques. This will be a valuable tool in enabling us to better understand the extent and roles of protein carbamylation in physiology.

Item Type:Thesis (Masters)
Award:Master of Science
Faculty and Department:Faculty of Science > Biological and Biomedical Sciences, School of
Thesis Date:2016
Copyright:Copyright of this thesis is held by the author
Deposited On:11 May 2016 10:36

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