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De novo design of knotted tandem repeat proteins.

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Takushi, Brittany 
Orozco, Carolina T 


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.


Acknowledgements: We thank Meredith Steward for assistance with biochemical experiments. Support for this work was provided by the Fred Hutchinson Cancer Center and by the National Institute for General Medical Sciences (NIGMS) for both BLS (R01 GM139752) and PB (R35 GM141457). L.F.M. was supported by a Human Frontier Science Program Cross Disciplinary Fellowship (LT000395/2020-C) and an EMBO Non-Stipendiary Fellowship (ALTF 1047-2019). Crystallographic data collection was conducted at the Advanced Light Source (ALS) at the Berkeley Center for Structural Biology which is supported in part by the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231. The Pilatus detector at Beamline 5.0.1 (where the data was collected) was funded under NIH grant S10OD021832. The ALS-ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169. SAXS data collection was also conducted at the Advanced Light Source, at the SYBLS beamline supported by NIH project ALS-ENABLE (P30 GM124169) and a High-End Instrumentation Grant S10 OD018483. Results shown in this report are derived from work performed at Structural Biology Center funded by the U.S. Department of Energy, Office of Biological and Environmental Research, and operated for the DOE Office of Science at the Advanced Photon Source by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.


Protein Conformation, Proteins, Protein Folding, Protein Domains, Tandem Repeat Sequences

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Nat Commun

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Springer Science and Business Media LLC
U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS) (R35 GM141457)