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

Abstract

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 ¹³ C‐ and ¹⁵ 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.more » Further work is therefore needed to assess the utility of MD simulation techniques for de novo protein design.« less

Authors:

Buchko, Garry W. ^[1]; Pulavarti, Surya V. S. R. K.  ^[2];

• Search DOE PAGES for author "Pulavarti, Surya V. S. R. K."
• Search DOE PAGES for ORCID "0000-0003-0888-9302"

Ovchinnikov, Victor ^[3]; Shaw, Elizabeth A. ^[2]; Rettie, Stephen A. ^[4]; Myler, Peter J. ^[5]; Karplus, Martin ^[6]; Szyperski, Thomas ^[2]; Baker, David ^[7]; Bahl, Christopher D.  ^[8]

• Search DOE PAGES for author "Bahl, Christopher D."
• Search DOE PAGES for ORCID "0000-0002-3652-3693"

---

+ Show Author Affiliations

1. Seattle Structural Genomics Center for Infectious Diseases Seattle Washington, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory Richland Washington 99352, School of Molecular Biosciences Washington State University Pullman Washington 99164
2. Department of Chemistry, State University of New York at Buffalo Buffalo New York 14260
3. Department of Chemistry and Chemical Biology Harvard University Cambridge Massachusetts
4. Institute for Protein Design University of Washington Seattle Washington 98195
5. Seattle Structural Genomics Center for Infectious Diseases Seattle Washington, Center for Infectious Disease Research Seattle Washington 98109, Department of Global Health University of Washington Seattle Washington 98165, Department of Biomedical Informatics and Health Education University of Washington Seattle Washington 98195
6. Department of Chemistry and Chemical Biology Harvard University Cambridge Massachusetts, Laboratoire de Chimie Biophysique, ISIS Universite de Strasbourg 67000 Strasbourg France
7. Institute for Protein Design University of Washington Seattle Washington 98195, Department of Biochemistry University of Washington Seattle Washington 98195, Howard Hughes Medical Institute, University of Washington Seattle Washington 98195
8. Institute for Protein Design University of Washington Seattle Washington 98195, Department of Biochemistry University of Washington Seattle Washington 98195, Institute for Protein Innovation Boston Massachusetts 02115

Publication Date:

Thu Oct 18 00:00:00 EDT 2018

Sponsoring Org.:

USDOE

OSTI Identifier:

1493377

Resource Type:

Publisher's Accepted Manuscript

Journal Name:

Protein Science

Additional Journal Information:

Journal Name: Protein Science Journal Volume: 27 Journal Issue: 9; Journal ID: ISSN 0961-8368

Publisher:

Wiley Blackwell (John Wiley & Sons)

Country of Publication:

United Kingdom

Language:

English