@article{Zhang2024,

title = {Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution},

author = {Jason Zhang, William Nguyen, Nathan Greenwood, John Rose, Shao-En Ong, Dustin Maly, David Baker},

url = {https://www.nature.com/articles/s41587-023-02107-w, Nature Biotechnology [Open Access]},

doi = {10.1038/s41587-023-02107-w},

year  = {2024},

date = {2024-01-25},

journal = {Nature Biotechnology},

abstract = {The utility of genetically encoded biosensors for sensing the activity of signaling proteins has been hampered by a lack of strategies for matching sensor sensitivity to the physiological concentration range of the target. Here we used computational protein design to generate intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity (Ras-LOCKR-S)) and proximity labelers of the Ras signaling environment (LOCKR-based, Ras activity-dependent Proximity Labeler (Ras-LOCKR-PL)). These tools allow the detection of endogenous Ras activity and labeling of the surrounding environment at subcellular resolution. Using these sensors in human cancer cell lines, we identified Ras-interacting proteins in oncogenic EML4-Alk granules and found that Src-Associated in Mitosis 68-kDa (SAM68) protein specifically enhances Ras activity in the granules. The ability to subcellularly localize endogenous Ras activity should deepen our understanding of Ras function in health and disease and may suggest potential therapeutic strategies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lajoie2020,

title = {Designed protein logic to target cells with precise combinations of surface antigens },

author = {Lajoie, Marc J. and

Boyken, Scott E. and

Salter, Alexander I. and

Bruffey, Jilliane and

Rajan, Anusha and

Langan, Robert A. and

Olshefsky, Audrey and

Muhunthan, Vishaka and

Bick, Matthew J. and

Gewe, Mesfin and

Quijano-Rubio, Alfredo and

Johnson, JayLee and

Lenz, Garreck and

Nguyen, Alisha and

Pun, Suzie and

Correnti, Colin E. and

Riddell, Stanley R. and

Baker, David},

url = {https://science.sciencemag.org/content/early/2020/08/19/science.aba6527

https://www.bakerlab.org/wp-content/uploads/2020/08/Lajoie-coLOCKR2020.pdf},

doi = {10.1126/science.aba6527},

year  = {2020},

date = {2020-08-20},

journal = {Science},

abstract = {Precise cell targeting is challenging because most mammalian cell types lack a single surface marker that distinguishes them from other cells. A solution would be to target cells based on specific combinations of proteins present on their surfaces. We design colocalization-dependent protein switches (Co-LOCKR) that perform AND, OR, and NOT Boolean logic operations. These switches activate through a conformational change only when all conditions are met, generating rapid, transcription-independent responses at single-cell resolution within complex cell populations. We implement AND gates to redirect T cell specificity against tumor cells expressing two surface antigens while avoiding off-target recognition of single-antigen cells, and 3-input switches that add NOT or OR logic to avoid or include cells expressing a third antigen. Thus, de novo designed proteins can perform computations on the surface of cells, integrating multiple distinct binding interactions into a single output.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kirkpatrick2020,

title = {Conditional Recruitment to a DNA-Bound CRISPR–Cas Complex Using a Colocalization-Dependent Protein Switch},

author = {Robin L. Kirkpatrick and Kieran Lewis and Robert A. Langan and Marc J. Lajoie and Scott E. Boyken and Madeleine Eakman and David Baker and Jesse G. Zalatan},

url = {https://pubs.acs.org/doi/full/10.1021/acssynbio.0c00012

https://www.bakerlab.org/wp-content/uploads/2020/08/Kirkpatrick2020-LOCKR-CRISPR.pdf},

doi = {10.1021/acssynbio.0c00012},

year  = {2020},

date = {2020-08-20},

journal = {ACS Synthetic Biology},

abstract = {To spatially control biochemical functions at specific sites within a genome, we have engineered a synthetic switch that activates when bound to its DNA target site. The system uses two CRISPR–Cas complexes to colocalize components of a de novo-designed protein switch (Co-LOCKR) to adjacent sites in the genome. Colocalization triggers a conformational change in the switch from an inactive closed state to an active open state with an exposed functional peptide. We prototype the system in yeast and demonstrate that DNA binding triggers activation of the switch, recruitment of a transcription factor, and expression of a downstream reporter gene. This DNA-triggered Co-LOCKR switch provides a platform to engineer sophisticated functions that should only be executed at a specific target site within the genome, with potential applications in a wide range of synthetic systems including epigenetic regulation, imaging, and genetic logic circuits.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ng2019,

title = {Modular and tunable biological feedback control using a de novo protein switch},

author = {Ng, Andrew H.

and Nguyen, Taylor H.

and Gómez-Schiavon, Mariana

and Dods, Galen

and Langan, Robert A.

and Boyken, Scott E.

and Samson, Jennifer A.

and Waldburger, Lucas M.

and Dueber, John E.

and Baker, David

and El-Samad, Hana},

url = {https://doi.org/10.1038/s41586-019-1425-7

https://www.nature.com/articles/s41586-019-1425-7

https://www.bakerlab.org/wp-content/uploads/2019/07/Ng_LOCKR_circuits.pdf},

doi = {10.1038/s41586-019-1425-7},

year  = {2019},

date = {2019-07-24},

journal = {Nature},

abstract = {De novo-designed proteins1–3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4–7. One such designer protein—degronLOCKR, which is based on ‘latching orthogonal cage–key proteins’ (LOCKR) technology8—is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}