期刊
出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2015037118
关键词
protein design; cryo-EM; DARPin; protein fusion; nanomaterials
资金
- National Institute of General Medical Sciences (NIGMS) under the NIH [T32GM008268]
- Open Philanthropy Project
- HHMI
- NSF [CHE-1629214]
- NIH [AI150464, HHSN272201700059C, P41GM128577]
- National Institute of Allergy and Infectious Diseases (NIAID) [DP1AI158186]
- NIGMS [R01GM120553]
- Pew Biomedical Scholars Award
- Burroughs Wellcome Investigators in the Pathogenesis of Infectious Diseases award
- Swiss National Science Foundation [310030_192689]
- Department of Energy (DOE) Office of Biological and Environmental Research
- NIH project ALS-ENABLE [P30 GM124169]
- NIH High-End Instrumentation Grant [S10OD018483]
- Human Frontiers Science Program Long Term Fellowship
- Washington Research Foundation
- Swiss National Science Foundation (SNF) [310030_192689] Funding Source: Swiss National Science Foundation (SNF)
Protein nanomaterial design using rigid fusion of homo-oligomer and spacer building blocks generates user-defined architectures with more geometric solutions, optimized using the Rosetta software. Experimental validation confirmed the assembly states for 11 designs mostly comprising designed ankyrin repeat proteins, exploring their use in cryo-EM structure determination. The dual anchoring strategy reduced flexibility in the target-DARPIN complex, suggesting potential for cryo-EM structure determination of small proteins.
Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via alpha-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 for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers 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 designed ankyrin repeat proteins (DARPins), held in place on one end by alpha-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, 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.
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