Sharma, Vangmayee (2017-07). The Engineering and Optimization of Pyrrolysyl-Synthetase as a Tool for Noncanonical Amino Acid Incorporation. Doctoral Dissertation.
Thesis
Since the discovery of pyrrolysine as the 22nd amino acid, the field of chemical biology has expanded tremendously with important developments made in genetic noncanonical amino acid (ncAA) incorporation based on the pyrrolysine incorporation machinery. As first discovered in methanogenic archaea, pyrrolysine is incorporated using pyrrolysyl-tRNA and the cognate pyrrolysyl-tRNA synthetase which install this amino acid using an in-frame stop codon present on the mRNA. This archaeal synthetase and tRNA form an orthogonal pair in naive organisms such as Escherichia coli, mammalian cells etc. and have been engineered over the past few years to incorporate several different noncanonical amino acids in proteins genetically. Although this system serves as an important chemical biology tool, it leaves much to be desired in terms of incorporation efficiency of these different unnatural amino acids. In this dissertation, we have explored the method for optimizing this remarkable system to achieve high levels of ncAA incorporation by engineering its N-terminal domain. Further, we then examined its application in improving other tRNA/synthetase systems which have been created for incorporation of specific ncAAs by researchers in previous years. In addition, we have also probed the feasibility of incorporating several histidine derivatives in proteins in using an engineered C-terminal domain version of this synthetase and the cognate tRNA to be able to study mechanism of enzymes employing histidines in their catalytic activity. In particular, the mechanism of alanine racemase enzyme was probed to validate the existence of a proton transfer chain near the catalytic site of the enzyme crucial to its activity. Overall, our results can be translated to other orthogonal tRNA/synthetase pairs derived from pyrrolysyl-tRNA/synthetase to achieve improvement in existing ncAA incorporation systems and hence resolve the poor incorporation problem of these ncAAs. Also, the system developed by us can be used to probe several enzymes which employ histidine triads in their catalytic mechanism. It can also be used to modify or fine-tune the activity of these enzymes without compromising protein structure.
ETD Chair
Liu, Wenshe Bovay Chair and Professor in Chemistry
