Biochemical investigation of carbamylated proteins from Arabidopsis thaliana

Carbon dioxide (CO2) plays a vital role in biological processes including photosynthesis, respiration, and multiple signalling pathways. CO2 is able to interact with proteins by forming carbamates on neutral amine groups. However, due to the labile nature of this post-translational modification, carbamates are extremely difficult to observe and have therefore gone under-researched. The recent development of a novel technique to trap carbamates on proteins has led to identification of a range of carbamylated proteins from Arabidopsis thaliana. This thesis describes an investigation into the effect of carbamylation on the activity of two such proteins. Fructose 1,6 bisphosphate aldolase (FBA1) is a metabolic enzyme that contains a carbamate modification on lysine 293. Activity assays conducted in the presence and absence of CO2 indicated that the cleavage activity of FBA1 is not affected by CO2 concentration. Similarly, analysis of subunit conformation indicated that the proportion of protein present as a tetramer was not affected by increasing the CO2 concentration. However, investigation of a mutant that cannot form a carbamate, FBA1-K293A, suggested that the carbamylated lysine residue may important for protein function. Mutating residue 293 to glutamate also produced a protein with reduced activity compared with the WT. It was not possible to conclude from trapping experiments and mass spectrometry analysis whether the carbamate modification may still have been present on the WT protein during earlier experiments in the absence of CO2. Three putative carbamates have previously been identified on the non-specific lipid binding protein LTP1. The lipid binding affinity of LTP1 was investigated using the fluorescent probe TNS. This was found to be significantly reduced in the absence of CO2. The mutant protein LTP1-K65A, which is unable to form one of the three putative carbamates identified on this protein, also had reduced binding in the absence of CO2. Preliminary dose response experiments indicated that this protein does have a lower affinity for TNS than the WT, however the effect observed in the absence of CO2 is likely to be due to the action of one of the other carbamates. Although the effect of carbamate modifications on the function of these two proteins was not fully determined in this investigation, the results suggest that the recently developed carbamate trapping technique provides an effective method for identifying functionally relevant novel carbamates.