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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01ht24wm91g
Title: Engineering and Unraveling Lasso Peptide Topologies
Authors: Allen, Caitlin D.
Advisors: Link, A. James
Contributors: Chemical and Biological Engineering Department
Keywords: Catenane
Lasso Peptide
Rotaxane
Subjects: Chemical engineering
Molecular biology
Chemistry
Issue Date: 2016
Publisher: Princeton, NJ : Princeton University
Abstract: Catenanes, rotaxanes and knots are characterized by their mechanically interlocked architectures. These molecules are of interest to scientists due to their rare and curious presence in nature, difficulty to synthesize, and potential for use in molecular machines. While synthetic organic chemists have made great strides in creating these entangled structures, very few examples exist in protein-based biological macromolecules. Lasso peptides are one of the only naturally occurring examples of a [1]rotaxane structure in protein space. These molecules are ribosomally synthesized and post-translationally modified into their unique threaded fold. Many of these peptides harbor native biological activity and impressive topological stability, and their potential use as a bioactive scaffold has motivated research in the field. While many approaches to engineer these molecules focus on genetic transformation, we show that altering the chemistry/shape of these structures in vitro offers a new route for creating and studying a wide range of topologically constrained and bioactive peptides. First, we broke down these structures to examine the thermal stability of lasso peptides. We studied the unthreading of astexin-2 and astexin-3 lasso peptide mutants using a combination of molecular dynamics and experimental techniques. We quantified activation energies and kinetics, and identified a new “tail pulling” mechanism to describe lasso peptide unfolding. Next, we edited lasso peptides with semi-synthetic methods. We cleaved open the loop of MccJ25 lasso peptide mutants using trypsin and chymotrypsin, transforming the [1]rotaxane to a [2]rotaxane. Following this, application of enzymatic or linker chemistry yielded a variety of [2]rotaxanes, as well as allowed for the insertion of new non-canonical chemical functionalities into the lasso peptide loop, recreating the [1]rotaxane. Finally, we created [3] and [4]catenanes using lasso peptides as a starting material. We mutated MccJ25 such that the loop can be cleaved, leaving a threaded strand flanked on both ends with cysteine. This allowed for site-specific disulfide conjugation of [2]rotaxanes to polymerize and cyclize, creating new, higher order catenanes, and we present the first structural NMR characterization of a solely peptide-based [3]catenane. We discuss the application of these lasso peptide-based structures to molecular machines, and gain some future perspectives on the field.
URI: http://arks.princeton.edu/ark:/88435/dsp01ht24wm91g
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: catalog.princeton.edu
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemical and Biological Engineering

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