Analysis of Thermostability of the Lasso Peptide Benenodin-1

Abstract

Lasso peptides are a class of ribosomally-synthesized and post-translationally-modified peptides (RiPPs). Recently, a new family of lasso peptides was discovered in Asticcacaulis benevestitus: benenodin. Of interest for this project is benenodin-1, a peptide lacking large “steric lock” residues typical for preventing lasso peptide unthreading. Instead, it contains two relatively small, polar residues, Glu14 and Gln15, which act as steric locks based on the NMR structure of the peptide. Previously, it was thought that ~60% of WT benenodin-1 samples unthread under heating at 95 °C, and that substitutions of the steric locks Glu14 to alanine and Gln15 to alanine show no unthreading behavior in the same assays. Further complicating the picture is the fact is that variants of Lys17 to alanine and Pro18 to alanine also demonstrate unthreading under heat. This thesis offers a new explanation for the unexplained benenodin-1 unthreading results. Namely, this work shows that most lasso peptide variants tested never fully unthread, and instead exist in one or two low-energy conformations after heating. This thesis examines the stability behavior of benenodin-1 using both computational and experimental techniques. Computationally, I model peptide unthreading for both thermolabile and thermostable variants using umbrella sampling. The protocol established for umbrella sampling in GROMACS is broadly applicable to umbrella sampling of other systems, and offers user-customizable parameters to ensure simulation success. In addition, I address shortcomings of biased simulations of the unthreading process, and offer an alternative approach. Experimentally, mutagenesis studies were used to elucidate the relative importance of different residues on the thermostability of benenodin-1. Carboxypeptidase assays show that most variants remain threaded in two dominant conformations after heating, and that these assays should be used in addition to existing isopeptidase assays for probing unthreading. Time-dependent evolution of the partially unthreaded species shows that partial unthreading is likely a reversible process. Together, these results show that lasso peptide thermostability can be coarsely modeled using computational methods, and add knowledge about partial unthreading that may support future development of scientific and medicinal applications of lasso peptides.