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Title: Investigating Lasso Peptide Unthreading Mechanisms in Astexin-3 Variants
Authors: Le, Dan Thanh Hoang
Advisors: Link, A. James
Department: Chemical and Biological Engineering
Class Year: 2015
Abstract: Lasso peptides are a class of natural products that are ribosomally synthesized and post-translationally modified. Their unique threaded lasso structure is typically held by two “steric lock” residues that prevent the tail from unthreading, but mutagenesis of these steric lock residues in astexin-3 results in mutants that can unthread when heat-treated for 3 hours at 95 °C. However, it is unknown how these lasso peptides unthread. In the case of tail-pulling, the C-terminus of the peptide is pulled until the loop region passes through the ring. In the case of loop-pulling, the loop region is pulled until the C-terminus passes through the ring. These mechanisms were investigated by pursuing a method of attaching the lasso peptides to relatively large magnetic beads via disulfide bonds using H\(_{2}\)O\(_{2}\). Both thermostable and thermolabile mutants that incorporate a cysteine residue at the Cterminus of astexin-3 were created. Mutants with the methionine residues replaced by leucine were also created to reduce the chances of oxidation. The unthreading behaviors of these mutants were characterized by HPLC and mass spectrometry. The leucine mutants were less thermostable than the peptides with methionine. Thermostable astexin-3 was reacted with the beads and H\(_{2}\)O\(_{2}\) in various buffers, and the beads were subsequently heated to examine the thermostability of the disulfide bonds between the peptides and the beads. It was found that most of the peptides detach from the beads, suggesting that the disulfide bonds are likely not thermostable at 95 °C. Investigations of the disulfide bridge can be later explored at other temperatures. A better understanding of these unthreading mechanisms will allow lasso peptides to be engineered for applications in the food and pharmaceutical packaging industries as molecular thermometers.
Extent: 47 pages
Type of Material: Princeton University Senior Theses
Language: en_US
Appears in Collections:Chemical and Biological Engineering, 1931-2017

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