Princeton University Library Catalog

The Role of Catalytic Residues in the Trans-Splicing Mechanism of the Nostoc punctiforme DnaE Split Intein

Author/​Artist:
Jalbut, Marla M. [Browse]
Format:
Senior thesis
Language:
English
Advisor(s):
Muir, Thomas [Browse]
Department:
Princeton University. Department of Chemistry [Browse]
Class year:
2013
Description:
82 pages
Restrictions note:
Walk-in Access. This thesis can only be viewed on computer terminals at the Mudd Manuscript Library.
Summary note:
Split inteins are fractured proteins that catalyze the splicing of two flanking polypeptides together through a trans-splicing reaction. This class of proteins has been the basis for a number of biotechnological applications that can be optimized through the engineering of fast-splicing inteins. Identifying and understanding the roles of catalytic residues in an intein sequence can aid in designing efficient split inteins. Interestingly, inteins in the fully functional cyanobacterial DnaE split intein family lack a histidine residue in block G that is highly conserved and essential for splicing activity in other inteins. In this study, I designed and tested several mutants with the aim of determining how the DnaE inteins and specifically Npu, which catalyzes particularly fast splicing reactions, overcome the lack of this key histidine residue. I sought to better characterize the roles of four residues in the Npu trans-splicing mechanism; these are residues that are either known to be or we postulated to be catalytic. I found that the serine at the position of the “missing” G-block histidine in Npu (S136) is not a major factor in this intein’s fast splicing activity, while the F-block aspartate and histidine residues both contribute to Npu’s fast splicing activity but are not the only factors helping Npu overcome its lack of the G-block histidine. Additionally, I showed for the first time that another conserved histidine, at position 48 in Npu, is important for both the initial steps of the reaction (branched intermediate formation) and even more so for completion of splicing (branched intermediate resolution). The results of this study provide a better understanding of key catalytic residues in the Npu splicing mechanism that can enable the engineering of inteins with desired splicing activities.