Student Theses and Dissertations

Neel H. Shah

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Muir Laboratory

Abstract

Inteins are auto-processing protein domains that carry out a post-translational process known as protein splicing. This process is characterized by excision of the intein (intervening protein) domain from within a larger polypeptide sequence with concomitant ligation of the flanking extein ( external protein) regions through a native peptide bond. Remarkably, a small subset of all inteins are naturally transcribed and translated as two fragments that efficiently associate and carry out the same biochemical process in trans, and these split inteins are potentially powerful tools for protein engineering. Recently, a split intein from the cyanobacterium Nostoc punctiforme (Npu) was discovered that can carry out protein splicing with a half-life of one minute, as opposed to hours as seen for previously characterized split and contiguous inteins. Inspired by the apparent uniqueness of this “ultrafast” splicing activity and its practical implications, we characterized several orthologous split inteins from the same family as Npu. Surprisingly, many of these inteins splice as quickly as Npu, and biochemical characterization of this family divulged sequence-activity correlations that provided insights into the molecular determinants for fast protein trans-splicing. Importantly, several of these inteins are extraordinarily efficient in their first auto-processing step, peptide bond cleavage coupled to thioester formation. Harnessing this property, along with efficient fragment association, a streamlined iteration of Expressed Protein Ligation (EPL), the most prevalent protein semi-synthesis technique, was developed. Further insights into protein splicing were obtained by the development of a novel kinetic assay that allowed for quantitative observation of a crucial intermediate in the protein splicing pathway, the branched intermediate (BI). Using this assay, BI resolution was unambiguously identified as the rate limiting step for Npu splicing. Furthermore, the roles of extein residues in individual steps along the splicing pathway were teased apart. Using protein semi-synthesis, kinetic measurements, and structural techniques, C-extein composition was found to be intimately linked to active-site dynamics and BI resolution kinetics. In addition to chemical reactivity, the fragment assembly of Npu was also characterized. Mutation of charged residues at the binding interface demonstrated that split intein binding affinity was dominated by intermolecular electrostatic interactions. By swapping charged residues between the intein fragments, a new split intein was engineered with orthogonal binding and reactivity to the wild-type Npu split intein. The wild-type and charges wapped inteins could be used in protein semi-synthesis endeavors requiring parallel selective splicing reactions in one pot. Finally, using a combination of biophysical techniques, the mechanism of split intein assembly was elucidated. Our analyses indicated that the assembly follows a unique trajectory comprised of coupled binding and folding of disordered regions of each fragment followed by a collapse of the structure into a stable functional domain. Collectively, these structural and functional studies not only provide insights into the inner workings of inteins but will also continue to aid in the development of important protein engineering technologies.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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