skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

Peptide drugs offer many advantages over traditional small-molecule therapeutics, including enhanced specificity and reduced off-target effects.  Constrained peptides are commonly employed by nature as signaling molecules or toxins, and the incorporation of multiple disulfide covalent crosslinks between pairs of cysteine residues is thought to be responsible for the stability and potent drug-like properties of these molecules.  Knottins and short-chain scorpion toxins are two well-characterized groups within this protein family; each is demarcated by conserved structural motifs and highly-conserved disulfide bonds. However, the ability to generate stable structures adopting a wide range of specified sizes and shapes not observed in nature will be necessary to unlock the full pharmacological potential of peptide-based drugs.  Working towards this goal, we previously reported a computational method for the de novo design of genetically encodable cysteine-rich peptides of various topologies, which were validated by experiments. Here, we describe two extensions to the above methodology. First, we present an efficient and generalizable method for the production of de novo designed, disulfide-rich peptides via genetic fusion to DsbC and expression in the cytoplasm of Escherichia coli.   This is demonstrated by producing two peptides with divergent structures and sequences: a mixed a/ß topology with a helix packing againstmore » a three-stranded antiparallel ß-sheet stabilized by three disulfide bonds (gHEEE_02), and a helix-turn-helix topology containing a single disulfide bond adjacent to the main-chain termini (gHH_02). The efficacy of the expression system to successfully fold peptides as designed was confirmed by determining the structure for these two peptides using solution NMR spectroscopy.  These two new NMR structures confirm the design accuracy for two additional de novo designed peptides that were not previously validated by full structural determination. Second, motivated by the observation that some of the computational designs did not appear to fold as expected, we developed a computational strategy based on molecular dynamics (MD) simulations to identify misfolded designs prior to synthesis and propose rescue mutations to improve these designs.« less

Authors:
ORCiD logo [1];  [2];  [3];  [4];  [4];  [5];  [3];  [2];  [4];  [4]
  1. BATTELLE (PACIFIC NW LAB)
  2. State University of New York at Buffalo
  3. Harvard University
  4. University of Washington
  5. Center for Infectious Disease Research
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1525972
Report Number(s):
PNNL-SA-129358
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Protein Science
Additional Journal Information:
Journal Volume: 27; Journal Issue: 9
Country of Publication:
United States
Language:
English
Subject:
structural biology, NMR spectroscopy, peptides, drug design, SSGCID

Citation Formats

Buchko, Garry W., Pulavarti, Surya V., Ovchinnikov, Victor, Shaw, Elizabeth A., Rettie, Stephan A., Myler, Peter J., Karplus, Martin, Szyperski, Thomas, Baker, David, and Bahl, Christopher D. Cytosolic expression, solution structures, and molecular dynamics simulation of genetically encodable disulfide-rich de novo designed peptides. United States: N. p., 2018. Web. doi:10.1002/pro.3453.
Buchko, Garry W., Pulavarti, Surya V., Ovchinnikov, Victor, Shaw, Elizabeth A., Rettie, Stephan A., Myler, Peter J., Karplus, Martin, Szyperski, Thomas, Baker, David, & Bahl, Christopher D. Cytosolic expression, solution structures, and molecular dynamics simulation of genetically encodable disulfide-rich de novo designed peptides. United States. doi:10.1002/pro.3453.
Buchko, Garry W., Pulavarti, Surya V., Ovchinnikov, Victor, Shaw, Elizabeth A., Rettie, Stephan A., Myler, Peter J., Karplus, Martin, Szyperski, Thomas, Baker, David, and Bahl, Christopher D. Sat . "Cytosolic expression, solution structures, and molecular dynamics simulation of genetically encodable disulfide-rich de novo designed peptides". United States. doi:10.1002/pro.3453.
@article{osti_1525972,
title = {Cytosolic expression, solution structures, and molecular dynamics simulation of genetically encodable disulfide-rich de novo designed peptides},
author = {Buchko, Garry W. and Pulavarti, Surya V. and Ovchinnikov, Victor and Shaw, Elizabeth A. and Rettie, Stephan A. and Myler, Peter J. and Karplus, Martin and Szyperski, Thomas and Baker, David and Bahl, Christopher D.},
abstractNote = {Peptide drugs offer many advantages over traditional small-molecule therapeutics, including enhanced specificity and reduced off-target effects.  Constrained peptides are commonly employed by nature as signaling molecules or toxins, and the incorporation of multiple disulfide covalent crosslinks between pairs of cysteine residues is thought to be responsible for the stability and potent drug-like properties of these molecules.  Knottins and short-chain scorpion toxins are two well-characterized groups within this protein family; each is demarcated by conserved structural motifs and highly-conserved disulfide bonds. However, the ability to generate stable structures adopting a wide range of specified sizes and shapes not observed in nature will be necessary to unlock the full pharmacological potential of peptide-based drugs.  Working towards this goal, we previously reported a computational method for the de novo design of genetically encodable cysteine-rich peptides of various topologies, which were validated by experiments. Here, we describe two extensions to the above methodology. First, we present an efficient and generalizable method for the production of de novo designed, disulfide-rich peptides via genetic fusion to DsbC and expression in the cytoplasm of Escherichia coli.   This is demonstrated by producing two peptides with divergent structures and sequences: a mixed a/ß topology with a helix packing against a three-stranded antiparallel ß-sheet stabilized by three disulfide bonds (gHEEE_02), and a helix-turn-helix topology containing a single disulfide bond adjacent to the main-chain termini (gHH_02). The efficacy of the expression system to successfully fold peptides as designed was confirmed by determining the structure for these two peptides using solution NMR spectroscopy.  These two new NMR structures confirm the design accuracy for two additional de novo designed peptides that were not previously validated by full structural determination. Second, motivated by the observation that some of the computational designs did not appear to fold as expected, we developed a computational strategy based on molecular dynamics (MD) simulations to identify misfolded designs prior to synthesis and propose rescue mutations to improve these designs.},
doi = {10.1002/pro.3453},
journal = {Protein Science},
number = 9,
volume = 27,
place = {United States},
year = {2018},
month = {9}
}

Works referenced in this record:

Enhanced sampling and applications in protein folding in explicit solvent
journal, June 2010

  • Zhang, Cheng; Ma, Jianpeng
  • The Journal of Chemical Physics, Vol. 132, Issue 24
  • DOI: 10.1063/1.3435332

Accurate de novo design of hyperstable constrained peptides
journal, September 2016

  • Bhardwaj, Gaurav; Mulligan, Vikram Khipple; Bahl, Christopher D.
  • Nature, Vol. 538, Issue 7625
  • DOI: 10.1038/nature19791

Processing of multi-dimensional NMR data with the new software PROSA
journal, November 1992

  • Güntert, Peter; Dötsch, Volker; Wider, Gerhard
  • Journal of Biomolecular NMR, Vol. 2, Issue 6
  • DOI: 10.1007/BF02192850

Protein NMR Structure Determination with Automated NOE Assignment Using the New Software CANDID and the Torsion Angle Dynamics Algorithm DYANA
journal, May 2002


New approach to Monte Carlo calculation of the free energy: Method of expanded ensembles
journal, February 1992

  • Lyubartsev, A. P.; Martsinovski, A. A.; Shevkunov, S. V.
  • The Journal of Chemical Physics, Vol. 96, Issue 3
  • DOI: 10.1063/1.462133

How to study proteins by circular dichroism
journal, August 2005

  • Kelly, Sharon M.; Jess, Thomas J.; Price, Nicholas C.
  • Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1751, Issue 2
  • DOI: 10.1016/j.bbapap.2005.06.005

A multi-pronged search for a common structural motif in the secretion signal of Salmonella enterica serovar Typhimurium type III effector proteins
journal, January 2010

  • Buchko, Garry W.; Niemann, George; Baker, Erin S.
  • Molecular BioSystems, Vol. 6, Issue 12
  • DOI: 10.1039/c0mb00097c

The program XEASY for computer-supported NMR spectral analysis of biological macromolecules
journal, July 1995

  • Bartels, Christian; Xia, Tai-he; Billeter, Martin
  • Journal of Biomolecular NMR, Vol. 6, Issue 1
  • DOI: 10.1007/BF00417486

Protein production by auto-induction in high-density shaking cultures
journal, May 2005


Protein NMR Recall, Precision, and F -measure Scores (RPF Scores):  Structure Quality Assessment Measures Based on Information Retrieval Statistics
journal, February 2005

  • Huang, Yuanpeng J.; Powers, Robert; Montelione, Gaetano T.
  • Journal of the American Chemical Society, Vol. 127, Issue 6
  • DOI: 10.1021/ja047109h

An overview of enzymatic reagents for the removal of affinity tags
journal, December 2011


Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes
journal, March 1977

  • Ryckaert, Jean-Paul; Ciccotti, Giovanni; Berendsen, Herman J. C.
  • Journal of Computational Physics, Vol. 23, Issue 3
  • DOI: 10.1016/0021-9991(77)90098-5

Crystallography & NMR System: A New Software Suite for Macromolecular Structure Determination
journal, September 1998

  • Brünger, A. T.; Adams, P. D.; Clore, G. M.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 54, Issue 5
  • DOI: 10.1107/S0907444998003254

Identification of Zinc-ligated Cysteine Residues Based on 13Cα and 13Cβ Chemical Shift Data
journal, April 2006

  • Kornhaber, Gregory J.; Snyder, David; Moseley, Hunter N. B.
  • Journal of Biomolecular NMR, Vol. 34, Issue 4
  • DOI: 10.1007/s10858-006-0027-5

Demonstrating an Order-of-Magnitude Sampling Enhancement in Molecular Dynamics Simulations of Complex Protein Systems
journal, February 2016

  • Pan, Albert C.; Weinreich, Thomas M.; Piana, Stefano
  • Journal of Chemical Theory and Computation, Vol. 12, Issue 3
  • DOI: 10.1021/acs.jctc.5b00913

An NMR approach to structural proteomics
journal, February 2002

  • Yee, A.; Chang, X.; Pineda-Lucena, A.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 4
  • DOI: 10.1073/pnas.042684599

Backbone and side chain 1H, 13C, and 15N NMR assignments for the organic hydroperoxide resistance protein (Ohr) from Burkholderia pseudomallei
journal, May 2009

  • Buchko, Garry W.; Hewitt, Stephen N.; Napuli, Alberto J.
  • Biomolecular NMR Assignments, Vol. 3, Issue 2
  • DOI: 10.1007/s12104-009-9165-5

Protein therapeutics: a summary and pharmacological classification
journal, January 2008

  • Leader, Benjamin; Baca, Quentin J.; Golan, David E.
  • Nature Reviews Drug Discovery, Vol. 7, Issue 1
  • DOI: 10.1038/nrd2399

Evaluating protein structures determined by structural genomics consortia
journal, December 2006

  • Bhattacharya, Aneerban; Tejero, Roberto; Montelione, Gaetano T.
  • Proteins: Structure, Function, and Bioinformatics, Vol. 66, Issue 4
  • DOI: 10.1002/prot.21165

Chemical shifts in proteins come of age
journal, January 1995


Simulated Tempering: A New Monte Carlo Scheme
journal, July 1992


Structure and folding of disulfide-rich miniproteins: Insights from molecular dynamics simulations and MM-PBSA free energy calculations
journal, April 2008

  • Combelles, Cecil; Gracy, Jérôme; Heitz, Annie
  • Proteins: Structure, Function, and Bioinformatics, Vol. 73, Issue 1
  • DOI: 10.1002/prot.22054

CHARMM: The biomolecular simulation program
journal, July 2009

  • Brooks, B. R.; Brooks, C. L.; Mackerell, A. D.
  • Journal of Computational Chemistry, Vol. 30, Issue 10
  • DOI: 10.1002/jcc.21287

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli
journal, January 2013

  • Nozach, Hervé; Fruchart-Gaillard, Carole; Fenaille, François
  • Microbial Cell Factories, Vol. 12, Issue 1
  • DOI: 10.1186/1475-2859-12-37

Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli
journal, October 2008

  • Qian, Zhi-Gang; Xia, Xiao-Xia; Choi, Jong Hyun
  • Biotechnology and Bioengineering, Vol. 101, Issue 3
  • DOI: 10.1002/bit.21898

Natural and Engineered Cystine Knot Miniproteins for Diagnostic and Therapeutic Applications
journal, December 2011


Global analysis of protein folding using massively parallel design, synthesis, and testing
journal, July 2017

  • Rocklin, Gabriel J.; Chidyausiku, Tamuka M.; Goreshnik, Inna
  • Science, Vol. 357, Issue 6347
  • DOI: 10.1126/science.aan0693

Screening, large-scale production and structure-based classification of cystine-dense peptides
journal, February 2018

  • Correnti, Colin E.; Gewe, Mesfin M.; Mehlin, Christopher
  • Nature Structural & Molecular Biology, Vol. 25, Issue 3
  • DOI: 10.1038/s41594-018-0033-9

Defining Solution Conformations of Small Linear Peptides
journal, June 1991


Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Refinement of protein structures in explicit solvent
journal, January 2003

  • Linge, Jens P.; Williams, Mark A.; Spronk, Christian A. E. M.
  • Proteins: Structure, Function, and Bioinformatics, Vol. 50, Issue 3
  • DOI: 10.1002/prot.10299

ACEMD: Accelerating Biomolecular Dynamics in the Microsecond Time Scale
journal, May 2009

  • Harvey, M. J.; Giupponi, G.; Fabritiis, G. De
  • Journal of Chemical Theory and Computation, Vol. 5, Issue 6
  • DOI: 10.1021/ct9000685

NMR with Proteins and Nucleic Acids
journal, January 1986


Torsion angle dynamics for NMR structure calculation with the new program Dyana
journal, October 1997

  • Güntert, P.; Mumenthaler, C.; Wüthrich, K.
  • Journal of Molecular Biology, Vol. 273, Issue 1
  • DOI: 10.1006/jmbi.1997.1284