De Novo Protein Design Using the Blueprint Builder in Rosetta

Abstract

While native proteins cover diverse structural spaces and achieve various biological events, not many of them can directly serve human needs. One reason is that the native proteins usually contain idiosyncrasies evolved for their native functions but disfavoring engineering requirements. To overcome this issue, one strategy is to create de novo proteins which are designed to possess improved stability, high environmental tolerance, and enhanced engineering potential. Compared to other protein engineering strategies, in silico design of de novo proteins has significantly expanded the protein structural and sequence spaces, reduced wet lab workload, and incorporated engineered features in a guided and efficient manner. In the Baker laboratory we have been applying a design pipeline that uses the blueprint builder to design different folds of de novo proteins, and have successfully obtained libraries of de novo proteins with improved stability and engineering potential. In this article, we will use the design of de novo β‐barrel proteins as an example to describe the principles and basic procedures of the blueprint builder−based design pipeline. © 2020 Wiley Periodicals LLC. Basic Protocol 1: The construction of blueprints Alternate Protocol: Build blueprints based on existing protein .pdb files Basic Protocol 2: De novo protein design pipeline using the blueprint builder

De novodesigned Hsp70 activator dissolves intracellular condensates

“…First 8 , we designed fully de novo JDMs that are predicted to bind Hsp70 at the same site as native JDs but are unrelated in structure and sequence. Second 9 , we built upon the two helices within DnaJ (a well-studied E. coli JDP) that interact with its partner (DnaK, the E. coli Hsp70) and added a third, designed helix to improve binding and monomer stability. In brief 8 , over a million designs were generated in silico and filtered based on their predicted binding to DnaK using Rosetta and AlphaFold2 (AF2) metrics (see Methods for details).…”

“…Miniprotein binder generation began by paring down J-domain by removing its first and fourth helix as they do not directly interact with DnaK. Afterwards, a third helix on top of the remaining helices were added by using the RosettaRemodel blueprint 9 program in order to increase stability of the remaining helices. 30,000 blueprints that differed in the lengths of the helices and the loop types were generated.…”

De novo design of protein homodimers containing tunable symmetric protein pockets

“…Repeat protein design as previously described ( 8) relies on Rosetta Monte Carlo fragment assembly approaches to explore repeat protein space (14). Unbiased sampling rarely yields repeat proteins with our desired helical parameters and only in cases where the lengths of the two helices in a repeat differ by 6-7 residues (SI Appendix, Fig.…”

Computer-aided discovery, design, and investigation of COVID-19 therapeutics

“…On the other hand, Cao et al applied two strategies for the design of mini-proteins to neutralize the SARS-CoV-2 spike protein RBD [ 70 ]. They firstly designed mini-protein incorporated with a derived helix of ACE2 (residues from 23 to 46) responsible for the interactions with the virus RBD by using the Rosetta blueprint builder [ 73 ]. They also de novo designed RBD-binding proteins by using rotamer interaction field docking [ 74 ] with large in silico mini-protein libraries [ 75 ], followed by the design to generate binders to the distinct regions of the RBD surface [ 70 ], the sequences of peptides designed are shown in Table 1 (AHB1-2, LCB1-8).…”