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Generation of ordered protein assemblies using rigid three-body fusion

Vulovic, Ivan and Yao, Qing and Park, Young-Jun and Courbet, Alexis and Norris, Andrew and Busch, Florian and Sahasrabuddhe, Aniruddha and Merten, Hannes and Sahtoe, Danny D. and Ueda, George and Fallas, Jorge A. and Weaver, Sara J. and Hsia, Yang and Langan, Robert A. and Pluckthun, Andreas and Wysocki, Vicki H. and Veesler, David and Jensen, Grant J. and Baker, David (2020) Generation of ordered protein assemblies using rigid three-body fusion. . (Unpublished)

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Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Vulovic, Ivan0000-0003-4208-6183
Yao, Qing0000-0003-3575-9909
Park, Young-Jun0000-0003-2901-6949
Courbet, Alexis0000-0003-0539-7011
Norris, Andrew0000-0002-0121-5922
Busch, Florian0000-0002-4324-6065
Sahasrabuddhe, Aniruddha0000-0002-3980-0035
Merten, Hannes0000-0003-2150-0867
Sahtoe, Danny D.0000-0001-9095-0744
Ueda, George0000-0002-9792-7149
Fallas, Jorge A.0000-0002-1431-820X
Weaver, Sara J.0000-0001-7753-6215
Hsia, Yang0000-0001-7467-8373
Langan, Robert A.0000-0003-3319-3016
Pluckthun, Andreas0000-0003-4191-5306
Wysocki, Vicki H.0000-0003-0495-2538
Veesler, David0000-0002-6019-8675
Jensen, Grant J.0000-0003-1556-4864
Baker, David0000-0001-7896-6217
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Posted July 19, 2020. Research reported in this publication was supported by the National Institute Of General Medical Sciences, by the National Institutes of Health under Award Number T32GM008268 to I.V., as well as the Open Philanthropy Project, Howard Hughes Medical Institute, and NSF Grant CHE-1629214 to D.B. NIH grant under award AI150464 provided support to G.J.J. Cryo-EM work under G.J.J. was performed in the Caltech Beckman Institute Resource Center for Transmission Electron Microscopy. We also thank Dr. Songye Chen and Dr. Andrey Malyutin at Caltech for technical assistance. This work was also supported by the NIAID / NIH (HHSN272201700059C to D.V.) NIGMS / NIH (R01GM120553 to D.V.), a Pew Biomedical Scholars Award to D.V., and a Burroughs Wellcome Investigators in the Pathogenesis of Infectious Diseases award to D.V. This work was also supported by NIH grant P41GM128577 to V.H.W and Swiss National Science Foundation grant 310030_192689 to A.P. In addition, we thank Kathryn Burnett and Greg Hura for SAXS data collection through the SIBYLS mail-in SAXS program at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported by DOE Office of Biological and Environmental Research. Additional support comes from the National Institute of Health project ALS-ENABLE (P30 GM124169) and a High-End Instrumentation Grant S10OD018483. A.C. is a recipient of the Human Frontiers Science Program Long Term Fellowship. A.C. and D.D.S. received Washington Research Foundation fellowships. We thank Albumedix for providing high quality Veltis-grade HSA. I.V. thanks Shane Caldwell (University of Washington) for discussion on SAXS analysis and Vikram Mulligan (Flatiron Institute) for general assistance with RosettaScripts.
Funding AgencyGrant Number
National Institute of General Medical SciencesUNSPECIFIED
Open Philanthropy ProjectUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Pew Charitable TrustUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Swiss National Science Foundation (SNSF)310030_192689
NIHP30 GM124169
Human Frontier Science ProgramUNSPECIFIED
Washington Research FoundationUNSPECIFIED
Record Number:CaltechAUTHORS:20200720-122329527
Persistent URL:
Official Citation:Generation of ordered protein assemblies using rigid three-body fusion. Ivan Vulovic, Qing Yao, Young-Jun Park, Alexis Courbet, Andrew Norris, Florian Busch, Aniruddha Sahasrabuddhe, Hannes Merten, Danny D Sahtoe, George Ueda, Jorge A Fallas, Yang Hsia, Sara J Weaver, Robert A Langan, Andreas Pluckthun, Vicki H Wysocki, David Veesler, Grant J Jensen, David Baker. bioRxiv 2020.07.18.210294; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104448
Deposited By: Tony Diaz
Deposited On:20 Jul 2020 21:11
Last Modified:20 Jul 2020 21:11

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