De novo design of bioactive protein switches

Abstract

Author Contributions RAL, AHN, and SEB contributed equally to this publication. RAL, SEB, ZC, WRPN, and DB conceived of the idea and initial steps for designing protein switches from de novo designed helical bundles. RAL and DB developed the thermodynamic model and the code upon which it works. RAL, SEB, and WRPN designed and biophysically characterized LOCKR scaffolds and BimLOCKR. RAL performed mutagenesis and Bio-layer interferometry experiments. SB characterized Bim interactions to Bcl2 homologs and aided experimental design. RAL performed design calculations for orthogonal LOCKR designs using code from SEB and VKM. AHN and RAL conceived of caging cODC. RAL performed design calculations to cage cODC and tune degronLOCKR. AHN conceived of and contributed to all experiments with degronLOCKR. THN performed dynamic measurement of degronLOCKR. AMW tested degronLOCKR in HEK293T cells. MJL, SEB, and RAL performed design calculations for asymmetric LOCKR. GD performed experiments with degronLOCKR and dCas9. GD contributed to plasmid and strain construction. RAL, SEB, and MJL conceived of caging sequences to control subcellular location and RAL performed design calculations for nesLOCKR. JAS and AHN performed all experiments for nesLOCKR. RAL, SEB, AHN, HE-S, and DB wrote the manuscript, all authors edited and approved.

Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

“…Specifically, the field is still lacking computational methods that will enable users to reliably design their system of choice without going through multiple time-consuming DBT cycles before arriving at the desired solution. Recent studies are emerging to tackle this challenge and to devise such methods for diverse RNA-, DNA-and protein-based applications, with varying degrees of success [1][2][3] . Perhaps the best known methods are the Cello algorithm and RBS calculator, which are limited to bacterial chassis 4,5 at the present time.…”

A bottom-up approach for thede novodesign of functional proteins

“…The design of novel functional proteins is a fundamental test to our understanding of protein structure and function, and has enormous potential in multiple arenas in basic biology and biotechnology (7)(8)(9)(10)(11)(12)(13)(14)(15)17). Nevertheless, the majority of studies where function was the main objective, existing proteins were repurposed and used as templates to install functional sites (7,(17)(18)(19)(20).…”

“…In contrast, the design of de novo proteins encoding biochemical functions is lagging far behind (6). Nonetheless, successes to date illustrate the potential of de novo design to transform multiple areas of biology and biotechnology, including the design of vaccine candidates (7-9), protein-based lead drugs (10), antivirals (11), pH-responsive carriers (12) and others (13)(14)(15).A widely used approach to design functional proteins is to transplant functional sites from their native context to heterologous proteins derived from the natural protein repertoire or de novo designed structures (16)(17)(18)(19). Commonly, we refer to this two-step approach, consisting of first selecting or building a stable, functionless scaffold, which subsequently serves as template for grafting, as a 'top-down' approach ( Fig.…”

“…In contrast, the design of de novo proteins encoding biochemical functions is lagging far behind (6). Nonetheless, successes to date illustrate the potential of de novo design to transform multiple areas of biology and biotechnology, including the design of vaccine candidates (7-9), protein-based lead drugs (10), antivirals (11), pH-responsive carriers (12) and others (13)(14)(15).…”

Computational design of a protein family that adopts two well-defined and structurally divergent de novo folds

“…In other cases, the engineered proteins have two well-defined states that are quite similar, for example the dynamic switching of DANCER proteins primarily involves a single tryptophan residue (Davey et al, 2017) , and the Rocker channel has two symmetrically related states that are structurally identical (Joh et al, 2017) . Finally, designed protein pH and peptide (Langan et al, 2019) dependent switches involve transitions between a single well-defined state and less well ordered states. Overall, in most previous work, the differences in conformation have either been relatively small, involved changes in oligomerization state, or only one of the two states has been well-defined.…”