Princeton University Library Catalog

Characterization of Thermal Lasso Peptide Unthreading in Astexins-2 and -3 Variants

Chen, Maria Yanqing [Browse]
Senior thesis
Link, A. James [Browse]
Princeton University. Department of Chemical and Biological Engineering [Browse]
Class year:
82 pages
Summary note:
Lasso peptides are ribosomally-synthesized and post-translationally modified peptides with threaded lasso structures, high proteolytic stability, and diverse bioactivities. Two sterically bulky amino acids on the threaded tail, the "steric lock", provide resistance to unthreading. Control of the unthreading behavior would unlock applications of lasso peptides as scaffolds for drug design and molecular thermometers. However, unthreading behavior has not been quantitatively characterized for any lasso peptide. This thesis provides a quantitative examination of lasso peptide unthreading and is the first to extensively study the unthreading of astexins-2 and -3. Astexins-2 and -3 are 24-residue lasso peptides found in the Gram-negative bacterium Asticcacaulis excentricus . Despite their similarities in sequence and structure, astexin-2 is temperature sensitive while astexin-3 is not. Through mutagenesis of the steric lock, I: 1) created thermostable astexin-2 variants and thermolabile astexin-3 variants and quantitatively characterized their unthreading kinetics, 2) illustrated that steric lock sequence confers a certain degree of, but does not completely dictate, thermostability in lasso peptides, 3) showed that drastic changes in the steric lock are not tolerated by astexins-2 and -3 biosynthetic machinery, and 4) found that cyclization and degradation of the precursor peptide may be competitive processes. Additionally, in collaboration with Andrew Ferguson at the University of Illinois at Urbana-Champaign, I performed molecular dynamics simulations that validated the use of umbrella sampling to map the free energy landscape of lasso peptide unthreading. The results of this study contribute to our understanding of the role of the steric lock in lasso peptide thermostability and will aid in the development of lasso peptides as products applicable to industry and basic research.