RESUMEN
Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.
Asunto(s)
Actinina/química , Células Epiteliales/metabolismo , Miosinas/química , Neuronas/metabolismo , Ingeniería de Proteínas/métodos , Espectrina/química , Actinina/genética , Actinina/metabolismo , Animales , Avena , Línea Celular , Chara , Pollos , Clonación Molecular , Dictyostelium , Células Epiteliales/citología , Células Epiteliales/efectos de la radiación , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Luz , Modelos Moleculares , Movimiento (Física) , Miosinas/genética , Miosinas/metabolismo , Neuronas/citología , Neuronas/efectos de la radiación , Óptica y Fotónica/métodos , Cultivo Primario de Células , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrina/genética , Espectrina/metabolismo , NicotianaRESUMEN
While deep learning models have seen increasing applications in protein science, few have been implemented for protein backbone generation-an important task in structure-based problems such as active site and interface design. We present a new approach to building class-specific backbones, using a variational auto-encoder to directly generate the 3D coordinates of immunoglobulins. Our model is torsion- and distance-aware, learns a high-resolution embedding of the dataset, and generates novel, high-quality structures compatible with existing design tools. We show that the Ig-VAE can be used with Rosetta to create a computational model of a SARS-CoV2-RBD binder via latent space sampling. We further demonstrate that the model's generative prior is a powerful tool for guiding computational protein design, motivating a new paradigm under which backbone design is solved as constrained optimization problem in the latent space of a generative model.
Asunto(s)
COVID-19 , ARN Viral , Humanos , Inmunoglobulinas , Proteínas/química , SARS-CoV-2RESUMEN
MOTIVATION: Recent advances in computational methods have facilitated large-scale sampling of protein structures, leading to breakthroughs in protein structural prediction and enabling de novo protein design. Establishing methods to identify candidate structures that can lead to native folds or designable structures remains a challenge, since few existing metrics capture high-level structural features such as architectures, folds and conformity to conserved structural motifs. Convolutional Neural Networks (CNNs) have been successfully used in semantic segmentation-a subfield of image classification in which a class label is predicted for every pixel. Here, we apply semantic segmentation to protein structures as a novel strategy for fold identification and structure quality assessment. RESULTS: We train a CNN that assigns each residue in a multi-domain protein to one of 38 architecture classes designated by the CATH database. Our model achieves a high per-residue accuracy of 90.8% on the test set (95.0% average per-class accuracy; 87.8% average per-structure accuracy). We demonstrate that individual class probabilities can be used as a metric that indicates the degree to which a randomly generated structure assumes a specific fold, as well as a metric that highlights non-conformative regions of a protein belonging to a known class. These capabilities yield a powerful tool for guiding structural sampling for both structural prediction and design. AVAILABILITY AND IMPLEMENTATION: The trained classifier network, parser network, and entropy calculation scripts are available for download at https://git.io/fp6bd, with detailed usage instructions provided at the download page. A step-by-step tutorial for setup is provided at https://goo.gl/e8GB2S. All Rosetta commands, RosettaRemodel blueprints, and predictions for all datasets used in the study are available in the Supplementary Information. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Redes Neurales de la Computación , Semántica , Bases de Datos Factuales , Proteínas , Programas InformáticosRESUMEN
RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-ß6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-ß6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.
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Sitio Alostérico/efectos de los fármacos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Proteínas ras/antagonistas & inhibidores , Proteínas ras/química , Animales , Anticuerpos Monoclonales/química , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Proteínas ras/metabolismoRESUMEN
Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.
Asunto(s)
Membranas Intracelulares/metabolismo , Fagosomas/metabolismo , Canales Catiónicos TRPC/metabolismo , Ácidos/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Diglicéridos/metabolismo , Exocitosis/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Humanos , Membranas Intracelulares/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Ratones , Viabilidad Microbiana/efectos de los fármacos , Modelos Biológicos , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , Fagosomas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Purinas/química , Purinas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Roscovitina , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Canal Catiónico TRPC6RESUMEN
The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrel fold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel protein has been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the fold remains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold) symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbone sampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequence design protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences are highly stable and fold to the designed barrel curvature as determined by a 2.1 Å resolution crystal structure. The designs show robustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in the hydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diverse hydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major step towards the de novo design of functional TIM barrels.