LINTHWAITE, VICTORIA,LOUISE (2017) Development of a Technology for the Discovery of Protein Carbamates. Doctoral thesis, Durham University.
|PDF - Accepted Version|
Carbon dioxide (CO2) is fundamental to life with critical roles in respiration, photosynthesis, metabolism, pathogenesis, and acid-base homeostasis. It is therefore remarkable that we know so little about the direct molecular interactions of CO2 with cellular components. CO2 is generally unreactive but combines rapidly with neutral amines at physiological temperatures and pressures to form carbamates. Carbamylation is caused by the nucleophilic attack of an uncharged amine (lysine side chain -amino group or N-terminal -amino group) on CO2. The carbamate modification has been observed on proteins including RuBisCO and haemoglobin but remains largely unexplored as a protein post-translational modification.
Carbamates are labile and previous work on this PTM has involved their study under non-physiological conditions. The objective of this thesis is to investigate this understudied modification by removing its labile nature through trapping of CO2 on its target proteins in conditions representative of a physiological environment.
This thesis presents a novel methodology to identify carbamates using a chemical trapping technique that eliminates their labile nature in combination with tryptic digest-MS analysis. The methodology functions under aqueous conditions representative of a physiological environment. Initial experiments demonstrated effective carbamate trapping at NH2 sites within the model substrates acetyl-lysine, PHE-GLY and PHE-LYS, a tetra-peptide and haemoglobin. The results were confirmed using ESI-MS combined with 12C and 13C isotope incorporation. Screening of Arabidopsis thaliana leaf lysates identified several novel carbamylated proteins previously unknown to directly interact with CO2. The proteomic screen was validated by the study of one new target, fructose bisphosphate aldolase 1, using recombinant protein.
This methodology provides a technology to identify sites of carbamate formation and will permit the identification of sites of CO2 interactions within proteomes. This research has produced a method capable of removing the labile nature of carbamates and thereby completely transforming the study of carbamylation as a PTM.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||21 Mar 2017 12:59|