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Title: Modular repeat protein sculpting using rigid helical junctions

Abstract

The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ~10-nm-diameter “L” and “V” shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales.

Authors:
ORCiD logo [1]; ORCiD logo [1];  [1];  [1];  [1]; ORCiD logo [1]
  1. Univ. of Washington, Seattle, WA (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
NIGMS; USDOE
OSTI Identifier:
1630336
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 117; Journal Issue: 16; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Brunette, TJ, Bick, Matthew J., Hansen, Jesse M., Chow, Cameron M., Kollman, Justin M., and Baker, David. Modular repeat protein sculpting using rigid helical junctions. United States: N. p., 2020. Web. https://doi.org/10.1073/pnas.1908768117.
Brunette, TJ, Bick, Matthew J., Hansen, Jesse M., Chow, Cameron M., Kollman, Justin M., & Baker, David. Modular repeat protein sculpting using rigid helical junctions. United States. https://doi.org/10.1073/pnas.1908768117
Brunette, TJ, Bick, Matthew J., Hansen, Jesse M., Chow, Cameron M., Kollman, Justin M., and Baker, David. Fri . "Modular repeat protein sculpting using rigid helical junctions". United States. https://doi.org/10.1073/pnas.1908768117. https://www.osti.gov/servlets/purl/1630336.
@article{osti_1630336,
title = {Modular repeat protein sculpting using rigid helical junctions},
author = {Brunette, TJ and Bick, Matthew J. and Hansen, Jesse M. and Chow, Cameron M. and Kollman, Justin M. and Baker, David},
abstractNote = {The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ~10-nm-diameter “L” and “V” shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales.},
doi = {10.1073/pnas.1908768117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 16,
volume = 117,
place = {United States},
year = {2020},
month = {4}
}

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