De novo design of knotted tandem repeat proteins

Doyle, Lindsey A.; Takushi, Brittany; Kibler, Ryan D.; Milles, Lukas F.; Orozco, Carolina T.; Jones, Jonathan D.; Jackson, Sophie E.; Stoddard, Barry L.; Bradley, Philip

doi:10.1038/s41467-023-42388-y

De novo design of knotted tandem repeat proteins

Journal Article · Tue Oct 24 00:00:00 EDT 2023 · Nature Communications

DOI:https://doi.org/10.1038/s41467-023-42388-y· OSTI ID:2471064

^[1]; Takushi, Brittany ^[1]; ^[2]; ^[2]; Orozco, Carolina T. ^[3]; ^[3]; ^[3]; ^[1]; ^[1]

Fred Hutchinson Cancer Center, Seattle, WA (United States)
University of Washington, Seattle, WA (United States)
University of Cambridge (United Kingdom)

De novo protein design methods can create proteins with folds not yet seen in nature. These methods largely focus on optimizing the compatibility between the designed sequence and the intended conformation, without explicit consideration of protein folding pathways. Deeply knotted proteins, whose topologies may introduce substantial barriers to folding, thus represent an interesting test case for protein design. Here we report our attempts to design proteins with trefoil (3₁) and pentafoil (5₁) knotted topologies. We extended previously described algorithms for tandem repeat protein design in order to construct deeply knotted backbones and matching designed repeat sequences (N = 3 repeats for the trefoil and N = 5 for the pentafoil). We confirmed the intended conformation for the trefoil design by X ray crystallography, and we report here on this protein’s structure, stability, and folding behaviour. The pentafoil design misfolded into an asymmetric structure (despite a 5-fold symmetric sequence); two of the four repeat-repeat units matched the designed backbone while the other two diverged to form local contacts, leading to a trefoil rather than pentafoil knotted topology. Our results also provide insights into the folding of knotted proteins.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); Human Frontier Science Program Cross Disciplinary Fellowship; EMBO Non-Stipendiary Fellowship; Fred Hutchinson Cancer Center; National Institute for General Medical Sciences (NIGMS)

Grant/Contract Number:: AC02-05CH11231; AC02-06CH11357

OSTI ID:: 2471064

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 14; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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