Cytosolic expression, solution structures, and molecular dynamics simulation of genetically encodable disulfide‐rich de novo designed peptides

Abstract

Disulfide-rich peptides represent an important protein family with broad pharmacological potential. Recent advances in computational methods have made it possible to design new peptides which adopt a stable conformation de novo. Here, we describe a system to produce disulfide-rich de novo peptides using Escherichia coli as the expression host. The advantage of this system is that it enables production of uniformly C- and N-labeled peptides for solution nuclear magnetic resonance (NMR) studies. This expression system was used to isotopically label two previously reported de novo designed peptides, and to determine their solution structures using NMR. The ensemble of NMR structures calculated for both peptides agreed well with the design models, further confirming the accuracy of the design protocol. Collection of NMR data on the peptides under reducing conditions revealed a dependency on disulfide bonds to maintain stability. Furthermore, we performed long-time molecular dynamics (MD) simulations with tempering to assess the stability of two families of de novo designed peptides. Initial designs which exhibited a stable structure during simulations were more likely to adopt a stable structure in vitro, but attempts to utilize this method to redesign unstable peptides to fold into a stable state were unsuccessful. Further work is therefore needed to assess the utility of MD simulation techniques for de novo protein design.

Structural diversity in the Mycobacteria DUF3349 superfamily

“…Note that while the sequence alignment in Figure suggests an α‐helix (α2) should exist between T24‐R35 in MAB_3043c, the NOE‐based structure calculations showed no evidence for a well‐defined α‐helix in this position. However, comparison of the 13 C α and 13 C β chemical shifts of MAB_3043c to random coil values does suggest some transient helical character between F28‐L33 (data not shown) . As will become clearer after describing the structure for MSMEG_1066, the stability of α2 might depend on the presence of α1.…”

“…Further biological experiments are necessary to answer these and other questions regarding the biological function of the DUF3349 family of proteins. Regardless of the answers to these biological questions, the solved structures serve to fill more “structure space” in the PDB and this assists bringing us better artificial intelligence‐based programs for predicting a protein's structures from its primary amino acid sequence and closer to being able to generate genetically encodable de novo designed proteins with targeted functions …”

“…Regardless of the answers to these biological questions, the solved structures serve to fill more "structure space" in the PDB and this assists bringing us better artificial intelligence-based programs for predicting a protein's structures from its primary amino acid sequence 33 and closer to being able to generate genetically encodable de novo designed proteins with targeted functions. 20,34 4 | EXPERIMENTAL PROCEDURES…”

“…However, comparison of the 13 C α and 13 C β chemical shifts of MAB_3043c to random coil values does suggest some transient helical character between F28-L33 (data not shown). 20 As will become clearer after describing the structure for MSMEG_1066, the stability of α2 might depend on the presence of α1.…”

Antifungal symbiotic peptide NCR044.1 exhibits unique structure and multi-faceted mechanisms of action that confer plant protection

“…and C15-C25. Note that the disulfide bonds are essential for the stability of the peptide's tertiary structure 21 as illustrated in the 1 H-15 N HSQC spectrum for reduced NCR044.1 in Fig. 2b.…”