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There has been considerable progress in the development of computational methods for designing protein-protein interactions, but engineering high-affinity binders without extensive screening and maturation remains challenging. Here, we test a protein design pipeline that uses iterative rounds of deep learning (DL)-based structure prediction (AlphaFold2) and sequence optimization (ProteinMPNN) to design autoinhibitory domains (AiDs) for a PD-L1 antagonist. Inspired by recent advances in therapeutic design, we sought to create autoinhibited (or masked) forms of the antagonist that can be conditionally activated by proteases. Twenty-threede novodesigned AiDs, varying in length and topology, were fused to the antagonist with a protease sensitive linker, and binding to PD-L1 was tested with and without protease treatment. Nine of the fusion proteins demonstrated conditional binding to PD-L1 and the top performing AiDs were selected for further characterization as single domain proteins. Without any experimental affinity maturation, four of the AiDs bind to the PD-L1 antagonist with equilibrium dissociation constants (KDs) below 150 nM, with the lowest KDequal to 0.9 nM. Our study demonstrates that DL-based protein modeling can be used to rapidly generate high affinity protein binders.Significance statementProtein-protein interactions are critical to most processes in biology, and improved methods for designing protein binders will enable the creation of new research reagents, diagnostics, and therapeutics. In this study, we show that a deep learning-based method for protein design can create high-affinity protein binders without the need for extensive screening or affinity maturation.

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Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

Many protein therapeutics are competitive inhibitors that function by binding to endogenous proteins and preventing them from interacting with native partners. One effective strategy for engineering competitive inhibitors is to graft structural motifs from a native partner into a host protein. Here, we develop and experimentally test a computational protocol for embedding binding motifs in de novo designed proteins. The protocol uses an “inside-out” approach: Starting with a structural model of the binding motif docked against the target protein, the de novo protein is built by growing new structural elements off the termini of the binding motif. During backbone assembly, a score function favors backbones that introduce new tertiary contacts within the designed protein and do not introduce clashes with the target binding partner. Final sequences are designed and optimized using the molecular modeling program Rosetta. To test our protocol, we designed small helical proteins to inhibit the interaction between Gαqand its effector PLC-β isozymes. Several of the designed proteins remain folded above 90°C and bind to Gαqwith equilibrium dissociation constants tighter than 80 nM. In cellular assays with oncogenic variants of Gαq, the designed proteins inhibit activation of PLC-β isozymes and Dbl-family RhoGEFs. Our results demonstrate that computational protein design, in combination with motif grafting, can be used to directly generate potent inhibitors without further optimization via high throughput screening or selection.statement for broader audienceEngineered proteins that bind to specific target proteins are useful as research reagents, diagnostics, and therapeutics. We used computational protein design to engineer de novo proteins that bind and competitively inhibit the G protein, Gαq, which is an oncogene for uveal melanomas. This computational method is a general approach that should be useful for designing competitive inhibitors against other proteins of interest.

Many proteins have low thermodynamic stability, which can lead to low expression yields and limit functionality in research, industrial and clinical settings. This article introduces two, web-based tools that use the high-resolution structure of a protein along with the Rosetta molecular modeling program to predict stabilizing mutations. The protocols were recently applied to three genetically and structurally distinct proteins and successfully predicted mutations that improved thermal stability and/or protein yield. In all three cases, combining the stabilizing mutations raised the protein unfolding temperatures by more than 20°C. The first protocol evaluates point mutations and can generate a site saturation mutagenesis heatmap. The second identifies mutation clusters around user-defined positions. Both applications only require a protein structure and are particularly valuable when a deep multiple sequence alignment is not available. These tools were created to simplify protein engineering and enable research that would otherwise be infeasible due to poor expression and stability of the native molecule.

Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of 16 modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications, such as protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a Web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels, as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, which binds the heterotrimeric G protein Gα(q.) The design is helical, well‐folded, and primarily monomeric in solution at a concentration of 10 μM. However, when we solved the crystal structure of SEWN0.1 at 1.9 Å, we observed a dimer in a conformation incompatible with binding Gα(q). Unintentionally, we had designed a protein that adopts alternate conformations depending on its oligomeric state. Recently, there has been tremendous progress in the field of protein structure prediction as new methods in artificial intelligence have been used to predict structures with high accuracy. We were curious if the structure prediction method AlphaFold could predict the structure of SEWN0.1 and if the prediction depended on oligomeric state. When AlphaFold was used to predict the structure of monomeric SEWN0.1, it produced a model that resembles the Rosetta design model and is compatible with binding Gα(q), but when used to predict the structure of a dimer, it predicted a conformation that closely resembles the SEWN0.1 crystal structure. AlphaFold's ability to predict multiple conformations for a single protein sequence should be useful for engineering protein switches.

The FastDesign protocol in the molecular modeling program Rosetta iterates between sequence optimization and structure refinement to stabilize de novo designed protein structures and complexes. FastDesign has been used previously to design novel protein folds and assemblies with important applications in research and medicine. To promote sampling of alternative conformations and sequences, FastDesign includes stages where the energy landscape is smoothened by reducing repulsive forces. Here, we discover that this process disfavors larger amino acids in the protein core because the protein compresses in the early stages of refinement. By testing alternative ramping strategies for the repulsive weight, we arrive at a scheme that produces lower energy designs with more native-like sequence composition in the protein core. We further validate the protocol by designing and experimentally characterizing over 4000 proteins and show that the new protocol produces higher stability proteins. This article is protected by copyright. All rights reserved.

Promiscuous enzymes that modify peptides and proteins are powerful tools for labeling biomolecules; however, directing these modifications to desired substrates can be challenging. Here, we use computational interface design to install a substrate recognition domain adjacent to the active site of a promiscuous enzyme, catechol O-methyltransferase. This design approach effectively decouples substrate recognition from the site of catalysis and promotes modification of peptides recognized by the recruitment domain. We determined the crystal structure of this novel multidomain enzyme, SH3-588, which shows that it closely matches our design. SH3-588 methylates directed peptides with catalytic efficiencies exceeding the wild-type enzyme by over 1,000-fold, whereas peptides lacking the directing recognition sequence do not display enhanced efficiencies. In competition experiments, the designer enzyme preferentially modifies directed substrates over undirected substrates, suggesting that we can use designed recruitment domains to direct post-translational modifications to specific sequence motifs on target proteins in complex multisubstrate environments.

Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.

Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, that binds the heterotrimeric G protein Gαq. The design is helical, well-folded, and primarily monomeric in solution at a concentration of 10 uM. However, when we solved the crystal structure of SEWN0.1, we observed a dimer in a conformation incompatible with binding Gαq. Unintentionally, we had designed a protein that adopts alternate conformations depending on its oligomeric state. Recently, there has been tremendous progress in the field of protein structure prediction as new methods in artificial intelligence have been used to predict structures with high accuracy. We were curious if the structure prediction method AlphaFold could predict the structure of SEWN0.1 and if the prediction depended on oligomeric state. When AlphaFold was used to predict the structure of monomeric SEWN0.1, it produced a model that resembles the Rosetta design model and is compatible with binding Gαq, but when used to predict the structure of a dimer, it predicted a conformation that closely resembles the SEWN0.1 crystal structure. AlphaFold’s ability to predict multiple conformations for a single protein sequence should be useful for engineering protein switches.

We report the measurement and analysis of sulfonium-π, thioether-π, and ammonium-π interactions in a β-hairpin peptide model system, coupled with computational investigation and PDB analysis. These studies indicated that the sulfonium-π interaction is the strongest and that polarizability contributes to the stronger interaction with sulfonium relative to ammonium. Computational studies demonstrate that differences in solvation of the trimethylsulfonium versus the trimethylammonium group also contribute to the stronger sulfonium-π interaction. In comparing sulfonium-π versus sulfur-π interactions in proteins, analysis of SAM- and SAH-bound enzymes in the PDB suggests that aromatic residues are enriched in close proximity to the sulfur of both SAM and SAH, but the populations of aromatic interactions of the two cofactors are not significantly different, with the exception of the Me-π interactions in SAM, which are the most prevalent interaction in SAM but are not possible for SAH. This suggests that the weaker interaction energies due to loss of the cation-π interaction in going from SAM to SAH may contribute to turnover of the cofactor.

Description, A stabilized dimer of the surface protein from dengue virus has been engineered to elicit antibodies that neutralize the virus., Dengue virus (DENV) is a worldwide health burden, and a safe vaccine is needed. Neutralizing antibodies bind to quaternary epitopes on DENV envelope (E) protein homodimers. However, recombinantly expressed soluble E proteins are monomers under vaccination conditions and do not present these quaternary epitopes, partly explaining their limited success as vaccine antigens. Using molecular modeling, we found DENV2 E protein mutations that induce dimerization at low concentrations (

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.

Protein engineering has contributed to successes in the field of T cell-based immunotherapy, including chimeric antigen receptor (CAR) T cell therapy. CAR T cell therapy has become a pillar of cancer immunotherapy, demonstrating clinical effectiveness against B cell malignancies by targeting the B cell antigen CD19. Current gene editing techniques have limited safety controls over CAR T cell activity, which presents a hurdle for control of CAR T cells in patients. Alternatively, CAR T cell activity can be controlled by engineering CARs to bind soluble adapter molecules that direct the interaction between the CAR T cell and target cell. The flexibility in this adapter-mediated approach overcomes the rigid specificity of traditional CAR T cells to allow targeting of multiple cell types. Here we describe adapter CAR T technologies and how these methods emphasize the growing role of protein engineering in the design of programmable tools for T cell therapies.

Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met. We describe the implementation of a test server framework and 40 scientific benchmarks, covering numerous applications in Rosetta bio-macromolecular modeling. High performance computing cluster integration allows these benchmarks to run continuously and automatically. Detailed protocol captures are useful for developers and users of Rosetta and other macromolecular modeling tools. The framework and design concepts presented here are valuable for developers and users of any type of scientific software and for the scientific community to create reproducible methods. Specific examples highlight the utility of this framework and the comprehensive documentation illustrates the ease of adding new tests in a matter of hours.

During signal transduction, the G protein, Gαq, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gαq, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gαq. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gαq with low micromolar affinity and inhibits Gαq by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gαq, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gαq, and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gαq/i with high affinity in vitro (KD = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gαq in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions.

Dengue viruses (DENV serotypes 1-4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging for the four DENV serotypes because of the need to achieve balanced replication of four independent vaccine components. Subunit vaccines represent an alternative approach that may circumvent problems inherent with live-attenuated DENV vaccines. In mature virus particles, the envelope (E) protein forms a homodimer that covers the surface of the virus and is the major target of neutralizing antibodies. Many neutralizing antibodies bind to quaternary epitopes that span across both E proteins in the homodimer. For soluble E (sE) protein to be a viable subunit vaccine, the antigens should be easy to produce and retain quaternary epitopes recognized by neutralizing antibodies. However, WT sE proteins are primarily monomeric at conditions relevant for vaccination and exhibit low expression yields. Previously, we identified amino acid mutations that stabilize the sE homodimer from DENV2 and dramatically raise expression yields. Here, we tested whether these same mutations raise the stability of sE from other DENV serotypes and ZIKV. We show that the mutations raise thermostability for sE from all the viruses, increase production yields from 4-fold to 250-fold, stabilize the homodimer, and promote binding to dimer-specific neutralizing antibodies. Our findings suggest that these sE variants could be valuable resources in the efforts to develop effective subunit vaccines for DENV serotypes 1 to 4 and ZIKV.

Engineered, light-sensitive protein switches are used to interrogate a broad variety of biological processes. These switches are typically constructed by genetically fusing naturally occurring light-responsive protein domains with functional domains from other proteins. Protein activity can be controlled using a variety of mechanisms including light-induced colocalization, caging, and allosteric regulation. Protein design efforts have focused on reducing background signaling, maximizing the change in activity upon light stimulation, and perturbing the kinetics of switching. It is common to combine structure-based modeling with experimental screening to identify ideal fusion points between domains and discover point mutations that optimize switching. Here, we introduce commonly used light-sensitive domains and summarize recent progress in using them to regulate protein activity.

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 A X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction.

Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers. IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.

The FastDesign protocol in the molecular modeling program Rosetta iterates between sequence optimization and structure refinement to stabilize de novo designed protein structures and complexes. FastDesign has been used previously to design novel protein folds and assemblies with important applications in research and medicine. To promote sampling of alternative conformations and sequences, FastDesign includes stages where the energy landscape is smoothened by reducing repulsive forces. Here, we discover that this process disfavors larger amino acids in the protein core because the protein compresses in the early stages of refinement. By testing alternative ramping strategies for the repulsive weight, we arrive at a scheme that produces lower energy designs with more native-like sequence composition in the protein core. We further validate the protocol by designing and experimentally characterizing over 4000 proteins and show that the new protocol produces higher stability proteins.

Many scientific disciplines rely on computational methods for data analysis, model generation, and prediction. Implementing these methods is often accomplished by researchers with domain expertise but without formal training in software engineering or computer science. This arrangement has led to underappreciation of sustainability and maintainability of scientific software tools developed in academic environments. Some software tools have avoided this fate, including the scientific library Rosetta. We use this software and its community as a case study to show how modern software development can be accomplished successfully, irrespective of subject area. Rosetta is one of the largest software suites for macromolecular modeling, with 3.1 million lines of code and many state-of-the-art applications. Since the mid 1990s, the software has been developed collaboratively by the RosettaCommons, a community of academics from over 60 institutions worldwide with diverse backgrounds including chemistry, biology, physiology, physics, engineering, mathematics, and computer science. Developing this software suite has provided us with more than two decades of experience in how to effectively develop advanced scientific software in a global community with hundreds of contributors. Here we illustrate the functioning of this development community by addressing technical aspects (like version control, testing, and maintenance), community-building strategies, diversity efforts, software dissemination, and user support. We demonstrate how modern computational research can thrive in a distributed collaborative community. The practices described here are independent of subject area and can be readily adopted by other software development communities.

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cβ) that increase the unfolding temperature of Cα/Cβ by 20 °C, improve the expression of four separate α/β TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cβ mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro., Recombinant T-cells receptors can redirect naïve T cells but often have low or uneven expression. Here, the authors model mutations in TCR constant domains to increase T cell receptor expression, assembly, and stability and generate bispecific molecules.

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light-activated nuclear shuttle (LANS) domain. Light activation resulted in efficient Set2-LANS nuclear localization followed by H3K36me3 deposition in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes showed disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs in a directed fashion on all transcribed genes regardless of their overall transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggests transcription-dependent and -independent mechanisms for H3K36me deposition and removal, respectively.

Background: Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. Results: We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light activated nuclear shuttle (LANS) domain. Early H3K36me3 dynamics identified rapid methylation in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes exhibited disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting a rate-limiting mechanism for H3K36me3 deposition. Removal of H3K36me3 was also rapid and highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Conclusion: Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggest transcription-dependent and independent mechanisms for H3K36me deposition and removal, respectively.