@article{nokey,

title = {Peptide-binding specificity prediction using fine-tuned protein structure prediction networks},

author = {Amir Motmaen, Justas Dauparas, Minkyung Baek, Mohamad H. Abedi, David Baker, Philip Bradley},

url = {https://www.pnas.org/doi/10.1073/pnas.2216697120, PNAS (Open Access)},

doi = {10.1073/pnas.2216697120},

year  = {2023},

date = {2023-02-21},

urldate = {2023-02-21},

journal = {Proceedings of the National Academy of Sciences},

abstract = {Peptide-binding proteins play key roles in biology, and predicting their binding specificity is a long-standing challenge. While considerable protein structural information is available, the most successful current methods use sequence information alone, in part because it has been a challenge to model the subtle structural changes accompanying sequence substitutions. Protein structure prediction networks such as AlphaFold model sequence-structure relationships very accurately, and we reasoned that if it were possible to specifically train such networks on binding data, more generalizable models could be created. We show that placing a classifier on top of the AlphaFold network and fine-tuning the combined network parameters for both classification and structure prediction accuracy leads to a model with strong generalizable performance on a wide range of Class I and Class II peptide-MHC interactions that approaches the overall performance of the state-of-the-art NetMHCpan sequence-based method. The peptide-MHC optimized model shows excellent performance in distinguishing binding and non-binding peptides to SH3 and PDZ domains. This ability to generalize well beyond the training set far exceeds that of sequence-only models and should be particularly powerful for systems where less experimental data are available.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Said2022,

title = {Exploration of Structured Symmetric Cyclic Peptides as Ligands for Metal-Organic Frameworks},

author = {Said, Meerit Y. and Kang, Christine S. and Wang, Shunzhi and Sheffler, William and Salveson, Patrick J. and Bera, Asim K. and Kang, Alex and Nguyen, Hannah and Ballard, Ryanne and Li, Xinting and Bai, Hua and Stewart, Lance and Levine, Paul and Baker, David},

url = {https://pubs.acs.org/doi/10.1021/acs.chemmater.2c02597, Chem. Mater.

https://www.bakerlab.org/wp-content/uploads/2022/10/Said_etal_ChemMater2022_CyclicPeptideMOFs.pdf, PDF},

doi = {/10.1021/acs.chemmater.2c02597},

year  = {2022},

date = {2022-10-25},

urldate = {2022-10-25},

journal = {Chemistry of Materials},

abstract = {Despite remarkable advances in the assembly of highly structured coordination polymers and metal–organic frameworks, the rational design of such materials using more conformationally flexible organic ligands such as peptides remains challenging. In an effort to make the design of such materials fully programmable, we first developed a computational design method for generating metal-mediated 3D frameworks using rigid and symmetric peptide macrocycles with metal-coordinating sidechains. We solved the structures of six crystalline networks involving conformationally constrained 6 to 12 residue cyclic peptides with C2, C3, and S2 internal symmetry and three different types of metals (Zn2+, Co2+, or Cu2+) by single-crystal X-ray diffraction, which reveals how the peptide sequences, backbone symmetries, and metal coordination preferences drive the assembly of the resulting structures. In contrast to smaller ligands, these peptides associate through peptide–peptide interactions without full coordination of the metals, contrary to one of the assumptions underlying our computational design method. The cyclic peptides are the largest peptidic ligands reported to form crystalline coordination polymers with transition metals to date, and while more work is required to develop methods for fully programming their crystal structures, the combination of high chemical diversity with synthetic accessibility makes them attractive building blocks for engineering a broader set of new crystalline materials for use in applications such as sensing, asymmetric catalysis, and chiral separation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Bhardwaj2022,

title = {Accurate de novo design of membrane-traversing macrocycles},

author = {Gaurav Bhardwaj and Jacob O’Connor and Stephen Rettie and Yen-Hua Huang and Theresa A. Ramelot and Vikram Khipple Mulligan and Gizem Gokce Alpkilic and Jonathan Palmer and Asim K. Bera and Matthew J. Bick and Maddalena {Di Piazza} and Xinting Li and Parisa Hosseinzadeh and Timothy W. Craven and Roberto Tejero and Anna Lauko and Ryan Choi and Calina Glynn and Linlin Dong and Robert Griffin and Wesley C. {van Voorhis} and Jose Rodriguez and Lance Stewart and Gaetano T. Montelione and David Craik and David Baker},

url = {https://www.sciencedirect.com/science/article/pii/S0092867422009229?via%3Dihub, Cell

https://www.bakerlab.org/wp-content/uploads/2022/08/1-s2.0-S0092867422009229-main.pdf, PDF},

doi = {10.1016/j.cell.2022.07.019},

year  = {2022},

date = {2022-08-29},

urldate = {2022-08-29},

journal = {Cell},

abstract = {We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6–12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6–12 residue size range cross membranes with an apparent permeability greater than 1 × 10−6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Macé2022,

title = {Cryo-EM structure of a type IV secretion system},

author = {Macé, Kévin

and Vadakkepat, Abhinav K.

and Redzej, Adam

and Lukoyanova, Natalya

and Oomen, Clasien

and Braun, Nathalie

and Ukleja, Marta

and Lu, Fang

and Costa, Tiago R. D.

and Orlova, Elena V.

and Baker, David

and Cong, Qian

and Waksman, Gabriel},

url = {https://www.nature.com/articles/s41586-022-04859-y, Nature

https://www.bakerlab.org/wp-content/uploads/2022/08/Mace2022s41586-022-04859-y.pdf, PDF},

doi = {10.1038/s41586-022-04859-y},

year  = {2022},

date = {2022-07-01},

urldate = {2022-07-01},

journal = {Nature},

abstract = {Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus—the conjugative type IV secretion system (T4SS)—produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament—the conjugative pilus—that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein–protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Generation of Potent and Stable GLP-1 Analogues Via “Serine Ligation”},

author = {Levine, Paul M. and Craven, Timothy W. and Li, Xinting and Balana, Aaron T. and Bird, Gregory H. and Godes, Marina and Salveson, Patrick J. and Erickson, Patrick W. and Lamb, Mila and Ahlrichs, Maggie and Murphy, Michael and Ogohara, Cassandra and Said, Meerit Y. and Walensky, Loren D. and Pratt, Matthew R. and Baker, David},

url = {https://pubs.acs.org/doi/abs/10.1021/acschembio.2c00075, ACS Chemical Biology

https://www.bakerlab.org/wp-content/uploads/2022/03/Levine_etal_ACSChemBio2022_GLP-1_ananlogues_by_serine_ligation.pdf, Download PDF},

doi = {10.1021/acschembio.2c00075},

year  = {2022},

date = {2022-03-23},

journal = {ACS Chemical Biology},

abstract = {Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of “serine ligation” for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.



},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Yao2022,

title = {De novo design and directed folding of disulfide-bridged peptide heterodimers},

author = {Yao, Sicong and Moyer, Adam and Zheng, Yiwu and Shen, Yang and Meng, Xiaoting and Yuan, Chong and Zhao, Yibing and Yao, Hongwei and Baker, David and Wu, Chuanliu},

url = {https://www.nature.com/articles/s41467-022-29210-x, Nature Communications

https://www.bakerlab.org/wp-content/uploads/2022/03/Yao_etal_NatComms2022_Design_of_directed_folding_of_disulfile_bridged_peptide_heterodimers.pdf, Download PDF},

year  = {2022},

date = {2022-03-22},

urldate = {2022-03-22},

journal = {Nature Communications},

abstract = {Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Computational methods have also been developed to design heterodimers of advanced functions. However, these peptide heterodimers are usually formed through noncovalent interactions, which are prone to dissociate and subject to concentration-dependent nonspecific aggregation. Heterodimers crosslinked with interchain disulfide bonds are more stable, but it represents a formidable challenge for both the computational design of heterodimers and the manipulation of disulfide pairing for heterodimer synthesis and applications. Here, we report the design, synthesis and application of interchain disulfide-bridged peptide heterodimers with mutual orthogonality by combining computational de novo designs with a directed disulfide pairing strategy. These heterodimers can be used as not only scaffolds for generating functional molecules but chemical tools or building blocks for protein labeling and construction of crosslinking hybrids. This study thus opens the door for using this unexplored dimeric structure space for many biological applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hosseinzadeh2021,

title = {Anchor extension: a structure-guided approach to design cyclic peptides targeting enzyme active sites},

author = {Hosseinzadeh, Parisa

and Watson, Paris R.

and Craven, Timothy W.

and Li, Xinting

and Rettie, Stephen

and Pardo-Avila, Fátima

and Bera, Asim K.

and Mulligan, Vikram Khipple

and Lu, Peilong

and Ford, Alexander S.

and Weitzner, Brian D.

and Stewart, Lance J.

and Moyer, Adam P.

and Di Piazza, Maddalena

and Whalen, Joshua G.

and Greisen, Per Jr.

and Christianson, David W.

and Baker, David},

url = {https://www.nature.com/articles/s41467-021-23609-8, Nature Communications

https://www.bakerlab.org/wp-content/uploads/2021/06/Hosseinzadeh_etal_NatureComms2021_AnchorExtention.pdf, Download PDF},

doi = {10.1038/s41467-021-23609-8},

year  = {2021},

date = {2021-06-07},

urldate = {2021-06-07},

journal = {Nature Communications},

abstract = {Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational “anchor extension” methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mulligan2021,

title = {Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1},

author = {Mulligan, Vikram Khipple and Workman, Sean and Sun, Tianjun and Rettie, Stephen and Li, Xinting and Worrall, Liam J. and Craven, Timothy W. and King, Dustin T. and Hosseinzadeh, Parisa and Watkins, Andrew M. and Renfrew, P. Douglas and Guffy, Sharon and Labonte, Jason W. and Moretti, Rocco and Bonneau, Richard and Strynadka, Natalie C. J. and Baker, David},

url = {https://www.pnas.org/content/118/12/e2012800118.full, PNAS

https://www.bakerlab.org/wp-content/uploads/2021/03/Mulligen_etal_PNAS2021_Macrocycle_inhibitors.pdf, Download PDF},

doi = {10.1073/pnas.2012800118},

year  = {2021},

date = {2021-03-23},

urldate = {2021-03-23},

journal = {Proceedings of the National Academy of Sciences},

volume = {118},

number = {12},

abstract = {The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of L- and D-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Quijano-Rubio2021,

title = {De novo design of modular and tunable protein biosensors},

author = {Quijano-Rubio, Alfredo

and Yeh, Hsien-Wei

and Park, Jooyoung

and Lee, Hansol

and Langan, Robert A.

and Boyken, Scott E.

and Lajoie, Marc J.

and Cao, Longxing

and Chow, Cameron M.

and Miranda, Marcos C.

and Wi, Jimin

and Hong, Hyo Jeong

and Stewart, Lance

and Oh, Byung-Ha

and Baker, David},

url = {https://www.nature.com/articles/s41586-021-03258-z, Nature

https://www.bakerlab.org/wp-content/uploads/2021/02/Rubio_et_al_Nature_COVID_LOCKR_sensors.pdf, Download PDF},

doi = {10.1038/s41586-021-03258-z},

year  = {2021},

date = {2021-01-27},

urldate = {2021-01-27},

journal = {Nature},

abstract = {Naturally occurring protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications1, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; binding of the analyte of interest drives switching from the closed to the open state. Because the sensor is based purely on thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the IgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-193, we used the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder4, has a limit of detection of 15 pM and a signal over background of over 50-fold. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mulligan2020,

title = {Computational design of mixed chirality peptide macrocycles with internal symmetry},

author = {Vikram Khipple Mulligan and Christine S. Kang and Michael R. Sawaya and Stephen Rettie and Xinting Li and Inna Antselovich and Timothy W. Craven and Andrew M. Watkins and Jason W. Labonte and Frank DiMaio and Todd O. Yeates and David Baker},

url = {https://onlinelibrary.wiley.com/doi/epdf/10.1002/pro.3974

https://www.bakerlab.org/wp-content/uploads/2020/10/Mulligan2020-Computational-design-of-mixed-chirality-peptide-macrocycles-with-internal-symmetry.pdf},

doi = {10.1002/pro.3974},

year  = {2020},

date = {2020-10-15},

journal = {Protein Science},

abstract = {Cyclic symmetry is frequent in protein and peptide homo‐oligomers, but extremely rare within a single chain, as it is not compatible with free N‐ and C‐termini. Here we describe the computational design of mixed‐chirality peptide macrocycles with rigid structures that feature internal cyclic symmetries or improper rotational symmetries inaccessible to natural proteins. Crystal structures of three C2‐ and C3‐symmetric macrocycles, and of six diverse S2‐symmetric macrocycles, match the computationally‐designed models with backbone heavy‐atom RMSD values of 1 å or better. Crystal structures of an S4‐symmetric macrocycle (consisting of a sequence and structure segment mirrored at each of three successive repeats) designed to bind zinc reveal a large‐scale zinc‐driven conformational change from an S4‐symmetric apo‐state to a nearly inverted S4‐symmetric holo‐state almost identical to the design model. This work demonstrates the power of computational design for exploring symmetries and structures not found in nature, and for creating synthetic switchable systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Xu2020,

title = {Computational design of transmembrane pores},

author = {Chunfu Xu and Peilong Lu and Tamer M. Gamal El-Din and Xue Y. Pei and Matthew C. Johnson and Atsuko Uyeda and Matthew J. Bick and Qi Xu and Daohua Jiang and Hua Bai and Gabriella Reggiano and Yang Hsia and T J Brunette and Jiayi Dou and Dan Ma and Eric M. Lynch and Scott E. Boyken and Po-Ssu Huang and Lance Stewart and Frank DiMaio and Justin M. Kollman and Ben F. Luisi and Tomoaki Matsuura and William A. Catterall and David Baker },

url = {https://www.bakerlab.org/wp-content/uploads/2020/08/Xuetal_Nature2020_DeNovoPores.pdf

https://www.nature.com/articles/s41586-020-2646-5},

doi = {10.1038/s41586-020-2646-5},

year  = {2020},

date = {2020-08-26},

journal = {Nature},

volume = {585},

pages = {129–134},

abstract = {Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels, and there have been recent advances in de novo membrane protein design and in redesigning naturally occurring channel-containing proteins. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore—but not the 12-helix pore—enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.},

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{Langan2019,

title = {De novo design of bioactive protein switches},

author = {Langan, Robert A.

 and Boyken, Scott E.

 and Ng, Andrew H.

 and Samson, Jennifer A.

 and Dods, Galen

 and Westbrook, Alexandra M.

 and Nguyen, Taylor H.

 and Lajoie, Marc J.

 and Chen, Zibo

 and Berger, Stephanie

 and Mulligan, Vikram Khipple

 and Dueber, John E.

 and Novak, Walter R. P.

 and El-Samad, Hana

 and Baker, David},

url = {https://doi.org/10.1038/s41586-019-1432-8

https://www.nature.com/articles/s41586-019-1432-8

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

doi = {10.1038/s41586-019-1432-8},

year  = {2019},

date = {2019-07-24},

journal = {Nature},

abstract = {Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix ‘cage’ with a single interface that can interact either intramolecularly with a terminal ‘latch’ helix or intermolecularly with a peptide ‘key’. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage–key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.},

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}

}

@article{Shen2018,

title = {De novo design of self-assembling helical protein filaments},

author = {Shen, Hao and Fallas, Jorge A. and Lynch, Eric and Sheffler, William and Parry, Bradley and Jannetty, Nicholas and Decarreau, Justin and Wagenbach, Michael and Vicente, Juan Jesus and Chen, Jiajun and Wang, Lei and Dowling, Quinton and Oberdorfer, Gustav and Stewart, Lance and Wordeman, Linda and De Yoreo, James and Jacobs-Wagner, Christine and Kollman, Justin and Baker, David},

url = {http://science.sciencemag.org/content/362/6415/705

https://www.bakerlab.org/wp-content/uploads/2018/12/Shen2018_filaments.pdf},

doi = {10.1126/science.aau3775},

issn = {0036-8075},

year  = {2018},

date = {2018-11-09},

journal = {Science},

volume = {362},

number = {6415},

pages = {705–709},

abstract = {There has been some success in designing stable peptide filaments; however, mimicking the reversible assembly of many natural protein filaments is challenging. Dynamic filaments usually comprise independently folded and asymmetric proteins and using such building blocks requires the design of multiple intermonomer interfaces. Shen et al. report the design of self-assembling helical filaments based on previously designed stable repeat proteins. The filaments are micron scale, and their diameter can be tuned by varying the number of repeats in the monomer. Anchor and capping units, built from monomers that lack an interaction interface, can be used to control assembly and disassembly.Science, this issue p. 705We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo{textendash}electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Geiger-Schuller2018,

title = {Extreme stability in de novo-designed repeat arrays is determined by unusually stable short-range interactions},

author = {Geiger-Schuller, Kathryn and Sforza, Kevin and Yuhas, Max and Parmeggiani, Fabio and Baker, David and Barrick, Doug},

url = {https://www.pnas.org/content/115/29/7539

https://www.bakerlab.org/wp-content/uploads/2019/02/Geiger-Schuller2018.pdf},

doi = {10.1073/pnas.1800283115},

issn = {0027-8424},

year  = {2018},

date = {2018-07-17},

journal = {PNAS},

volume = {115},

number = {29},

pages = {7539-7544},

abstract = {We apply a statistical thermodynamic formalism to quantify the cooperativity of folding of de novo-designed helical repeat proteins (DHRs). This analysis provides a fundamental thermodynamic description of folding for de novo-designed proteins and permits comparison with naturally occurring repeat protein thermodynamics. We find that individual DHR units are intrinsically stable, unlike those of naturally occurring proteins. This observation reveals local (intrarepeat) interactions as a source of high stability in Rosetta-designed proteins and suggests that different types of DHR repeats may be combined in a single polypeptide chain, expanding the repertoire of folded DHRs for applications such as molecular recognition. Favorable intrinsic stability imparts a downhill shape to the energy landscape, suggesting that DHRs fold fast and through parallel pathways.Designed helical repeats (DHRs) are modular helix{textendash}loop{textendash}helix{textendash}loop protein structures that are tandemly repeated to form a superhelical array. Structures combining tandem DHRs demonstrate a wide range of molecular geometries, many of which are not observed in nature. Understanding cooperativity of DHR proteins provides insight into the molecular origins of Rosetta-based protein design hyperstability and facilitates comparison of energy distributions in artificial and naturally occurring protein folds. Here, we use a nearest-neighbor Ising model to quantify the intrinsic and interfacial free energies of four different DHRs. We measure the folding free energies of constructs with varying numbers of internal and terminal capping repeats for four different DHR folds, using guanidine-HCl and glycerol as destabilizing and solubilizing cosolvents. One-dimensional Ising analysis of these series reveals that, although interrepeat coupling energies are within the range seen for naturally occurring repeat proteins, the individual repeats of DHR proteins are intrinsically stable. This favorable intrinsic stability, which has not been observed for naturally occurring repeat proteins, adds to stabilizing interfaces, resulting in extraordinarily high stability. Stable repeats also impart a downhill shape to the energy landscape for DHR folding. These intrinsic stability differences suggest that part of the success of Rosetta-based design results from capturing favorable local interactions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hosseinzadeh2017,

title = {Comprehensive computational design of ordered peptide macrocycles},

author = {Hosseinzadeh, Parisa* and Bhardwaj, Gaurav* and Mulligan, Vikram Khipple* and Shortridge, Matthew D. and Craven, Timothy W. and Pardo-Avila, F{'a}tima and Rettie, Stephen A. and Kim, David E. and Silva, Daniel-Adriano and Ibrahim, Yehia M. and Webb, Ian K. and Cort, John R. and Adkins, Joshua N. and Varani, Gabriele and Baker, David},

url = {http://science.sciencemag.org/content/358/6369/1461

https://www.bakerlab.org/wp-content/uploads/2017/12/Science_Hosseinzadeh_et_al_2017.pdf},

doi = {10.1126/science.aap7577},

issn = {0036-8075},

year  = {2017},

date = {2017-12-15},

journal = {Science},

volume = {358},

number = {6369},

pages = {1461-1466},

abstract = {Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Butterfield2017,

title = {Evolution of a designed protein assembly encapsulating its own RNA genome},

author = {Butterfield, Gabriel L.*

and Lajoie, Marc J.*

and Gustafson, Heather H.

and Sellers, Drew L.

and Nattermann, Una

and Ellis, Daniel

and Bale, Jacob B.

and Ke, Sharon

and Lenz, Garreck H.

and Yehdego, Angelica

and Ravichandran, Rashmi

and Pun, Suzie H.

and King, Neil P.

and Baker, David},

url = {http://dx.doi.org/10.1038/nature25157

https://www.bakerlab.org/wp-content/uploads/2017/12/Nature_Butterfield_et_al_2017.pdf},

doi = {10.1038/nature25157},

issn = {1476-4687},

year  = {2017},

date = {2017-12-13},

journal = {Nature},

abstract = {The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6hours of treatment), and in vivo circulation time (from less than 5minutes to approximately 4.5hours). The resulting synthetic nucleocapsids package one full length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at ‘top-down’ modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary ‘bottom-up’ approach with considerable advantages in programmability and control.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Bhardwaj2016,

title = {Accurate de novo design of hyperstable constrained peptides},

author = { Gaurav Bhardwaj* and Vikram Khipple Mulligan* and Christopher D. Bahl* and Jason M. Gilmore and Peta J. Harvey and Olivier Cheneval and Garry W. Buchko and Surya V. S. R. K. Pulavarti and Quentin Kaas and Alexander Eletsky and Po-Ssu Huang and William A. Johnsen and Per Jr Greisen and Gabriel J. Rocklin and Yifan Song and Thomas W. Linsky and Andrew Watkins and Stephen A. Rettie and Xianzhong Xu,	Lauren P. Carter and Richard Bonneau and James M. Olson and Evangelos Coutsias and Colin E. Correnti and Thomas Szyperski and David J. Craik and David Baker	},

url = {https://www.bakerlab.org/wp-content/uploads/2016/09/Bhardwaj_Nature_2016.pdf},

doi = {10.1038/nature19791},

year  = {2016},

date = {2016-09-14},

journal = {Nature},

abstract = {Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18–47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N–C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{L2015,

title = {Rational design of α-helical tandem repeat proteins with closed architectures},

author = {L Doyle and J Hallinan and J Bolduc and F Parmeggiani and D Baker and BL Stoddard and P Bradley},

url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Doyle_Nature_2015.pdf},

doi = {10.1038/nature16191},

year  = {2015},

date = {2015-12-24},

journal = {Nature},

volume = {528(7583)},

pages = {585-8},

abstract = {Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. The overall architecture of tandem repeat protein structures--which is dictated by the internal geometry and local packing of the repeat building blocks--is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed α-solenoid repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed α-solenoid repeats with a left-handed helical architecture that--to our knowledge--is not yet present in the protein structure database.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}