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| Open AccessStimulus-responsive assembly of nonviral nucleocapsids
A stimulus-responsive approach for recapitulating nonviral nucleocapsid assembly on demand under controlled conditions provides a robust platform for applications in synthetic biology and mRNA nanomedicine.
- Mao Hori
- , Angela Steinauer
- & Donald Hilvert
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Article
| Open AccessLocal environment in biomolecular condensates modulates enzymatic activity across length scales
Here, the authors show that biomolecular condensates can enhance enzymatic rates by creating distinct solvent environments compared to the surrounding solution, and this emergent property can manifest within assemblies as small as nanometers.
- Marcos Gil-Garcia
- , Ana I. Benítez-Mateos
- & Paolo Arosio
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| Open AccessA dual diffusion model enables 3D molecule generation and lead optimization based on target pockets
Structure-based generative chemistry is crucial in computer-aided drug discovery. Here, authors propose PMDM, a conditional generative model for 3D molecule generation tailored to specific targets. Extensive experiments demonstrate that PMDM can effectively generate rational bioactive molecules
- Lei Huang
- , Tingyang Xu
- & Hengtong Zhang
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Article
| Open AccessMachine learning-aided design and screening of an emergent protein function in synthetic cells
Here, the authors introduce a pipeline to screen machine learning generated variants of a protein that forms intracellular spatiotemporal patterns in E. coli, demonstrating the best variants can substitute the wildtype gene.
- Shunshi Kohyama
- , Béla P. Frohn
- & Petra Schwille
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| Open AccessProtein design using structure-based residue preferences
Recent protein design methods rely on large neural networks, yet it is unclear which dependencies are critical for determining function. Here, authors show that learning the per residue mutation preferences, without considering interactions, enables design of functional and diverse protein variants.
- David Ding
- , Ada Y. Shaw
- & Debora S. Marks
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| Open AccessDesign of target specific peptide inhibitors using generative deep learning and molecular dynamics simulations
Here the authors report a computational approach which integrates deep learning and structural modelling to design target-specific peptides. They apply this to β-catenin and NF-κB essential modulator, resulting in improved binding, highlighting the efficacy of this strategy.
- Sijie Chen
- , Tong Lin
- & Xiaolin Cheng
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| Open AccessA system for inducible mitochondria-specific protein degradation in vivo
Conditional degradation of proteins is instrumental to advance our knowledge of cell biology but has been lacking for organelles like mitochondria. Here, the authors develop a proteolysis system based on the mycoplasma Lon protease that functions selectively within mitochondria in yeast and human cells.
- Swastika Sanyal
- , Anna Kouznetsova
- & Camilla Björkegren
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Article
| Open AccessA general computational design strategy for stabilizing viral class I fusion proteins
The authors present a generalisable computational approach to stabilize class I fusion proteins in the prefusion state. The method was used to stabilize the fusion proteins of RSV, hMPV, and SARS-CoV-2 viruses, with the designs structurally validated and RSV F protein assessed in a neutralization assay.
- Karen J. Gonzalez
- , Jiachen Huang
- & Eva-Maria Strauch
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| Open AccessAntiviral fibrils of self-assembled peptides with tunable compositions
In this work, the authors report the use of a computationally and rationally designed self-assembling peptide that has robust antiviral capability with demonstrated specificity in binding to SARS-CoV-2 and inhibition of viral entry into human cells.
- Joseph Dodd-o
- , Abhishek Roy
- & Vivek Kumar
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Article
| Open AccessMAIVeSS: streamlined selection of antigenically matched, high-yield viruses for seasonal influenza vaccine production
Vaccines combat global influenza threats, relying on timely selection of optimal seed viruses. Here, authors introduce MAIVeSS, a machine learning assisted framework to streamline vaccine seed virus selection using genomic sequence, expediting seasonal flu vaccine production and supply.
- Cheng Gao
- , Feng Wen
- & Xiu-Feng Wan
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| Open AccessProtein structure generation via folding diffusion
The ability to engineer novel protein structures has tremendous scientific and therapeutic impact. Here, authors develop a generative model acting upon an angular representation of protein structures to create high quality protein backbones.
- Kevin E. Wu
- , Kevin K. Yang
- & Ava P. Amini
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Article
| Open AccessProgrammable RNA base editing with photoactivatable CRISPR-Cas13
Cas13 systems suffer from a lack of spatiotemporal control. Here the authors report paCas13, a light-inducible Cas13 system created by fusing Magnet with fragment pairs; they also report padCas13, a light-inducible base-editing system by fusing ADAR2 to catalytically inactive paCas13 fragments.
- Jeonghye Yu
- , Jongpil Shin
- & Won Do Heo
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Article
| Open Accessi-shaped antibody engineering enables conformational tuning of biotherapeutic receptor agonists
In contrast to their clinical success as inhibitors and targeting agents, antibodies have generally been ineffective as receptor agonists. Here, Romei et al. leverage a natural homotypic interface to tune antibody geometry, enabling optimization of agonist activity for multiple therapeutic targets.
- Matthew G. Romei
- , Brandon Leonard
- & Greg A. Lazar
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| Open AccessACIDES: on-line monitoring of forward genetic screens for protein engineering
Screening mutated proteins is a versatile strategy in protein research, producing massive datasets when combined with NGS. Here, authors present ACIDES to estimate mutated protein fitness and aid protein engineering pipelines in a range of applications, including gene therapy.
- Takahiro Nemoto
- , Tommaso Ocari
- & Ulisse Ferrari
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Article
| Open AccessOptimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors
Designing optogenetically functionalized G protein-coupled receptors (optoXRs) to mimic endogenous signaling in vivo is challenging. Here, the authors optimize the design of optoXRs by considering evolutionary conserved protein interactions, and they employ this approach in fruit flies.
- Fangmin Zhou
- , Alexandra-Madelaine Tichy
- & Peter Soba
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| Open AccessDirecting polymorph specific calcium carbonate formation with de novo protein templates
Most proteins mediating biomineralization in nature are not well structured, and the structures of the relevant protein-mineral interfaces regulating mineralization are elusive. Here, the authors computationally design proteins that modulate calcium carbonate mineralization to generate hybrid materials and elucidate the roles of designed proteins in controlling mineralization.
- Fatima A. Davila-Hernandez
- , Biao Jin
- & David Baker
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| Open AccessA computational toolbox for the assembly yield of complex and heterogeneous structures
Predicting the effective assembly of a set of proteins into a desired structure has traditionally been a challenging task. Here, authors demonstrate that advancements in automatic differentiation make it possible to address this problem using classical statistical mechanics.
- Agnese I. Curatolo
- , Ofer Kimchi
- & Michael P. Brenner
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Article
| Open AccessUniKP: a unified framework for the prediction of enzyme kinetic parameters
Prediction of enzyme kinetic parameters is essential for designing and optimising enzymes for various biotechnological and industrial applications. Here, authors presented a prediction framework (UniKP), which improves the accuracy of predictions for three enzyme kinetic parameters.
- Han Yu
- , Huaxiang Deng
- & Xiaozhou Luo
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Article
| Open AccessDesign and structural validation of peptide–drug conjugate ligands of the kappa-opioid receptor
Despite advances in GPCR structures and peptide design, creating high-affinity ligands remains a challenge. Here the authors develop a computational method, successfully identifying peptide-based molecules for KOR: their platform shows promise for streamlined GPCR ligand discovery.
- Edin Muratspahić
- , Kristine Deibler
- & Christian W. Gruber
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| Open AccessProgrammable de novo designed coiled coil-mediated phase separation in mammalian cells
Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. Here the authors design protein liquid condensates from tunable concatenated coiled-coil dimer modules, unraveling the principles for coexisting condensates, chemical regulation, formation from either one or two polypeptide components in mammalian cells.
- Maruša Ramšak
- , Dominique A. Ramirez
- & Roman Jerala
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| Open AccessEngineered immunogens to elicit antibodies against conserved coronavirus epitopes
A pan-betacoronavirus vaccine will likely require the elicitation of antibodies against spike regions conserved across diverse coronaviruses. Here, authors computationally engineer and experimentally validate immunogens to elicit antibodies against two such spike regions.
- A. Brenda Kapingidza
- , Daniel J. Marston
- & Mihai L. Azoitei
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| Open AccessUnexpected dynamics in femtomolar complexes of binding proteins with peptides
High binding affinities are usually obtained when ligands are rigidified. Here the authors present flexible peptides binding to Armadillo repeat proteins with femtomolar affinity. They demonstrate that the bound state is characterized by residual dynamics limiting entropic losses upon binding.
- Stefano Cucuzza
- , Malgorzata Sitnik
- & Oliver Zerbe
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| Open AccessProRefiner: an entropy-based refining strategy for inverse protein folding with global graph attention
Inverse Protein Folding is a critical component of protein design. Here, authors introduce ProRefiner, a deep-learning model for IPF that exhibits both high performance and memory efficiency, thereby contributing to advancements in protein design.
- Xinyi Zhou
- , Guangyong Chen
- & Pheng Ann Heng
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| Open AccessSimulation-guided engineering of split GFPs with efficient β-strand photodissociation
Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live cell imaging. Here, authors have used computational methods to engineer a fast-dissociating split GFP, which could be used to study macromolecular interactions.
- Yasmin Shamsudin
- , Alice R. Walker
- & Steven G. Boxer
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| Open AccessRemodeling the cellular stress response for enhanced genetic code expansion in mammalian cells
Genetic code expansion (GCE) is a protein engineering tool that enables programmed and site-specific installation of noncanonical amino acids into proteins. Here, authors show that cellular stress remodelling boosts GCE in mammalian cells including GCE realized by orthogonally translating organelles.
- Mikhail E. Sushkin
- , Christine Koehler
- & Edward A. Lemke
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Article
| Open AccessLactate biosensors for spectrally and spatially multiplexed fluorescence imaging
l-Lactate is increasingly recognized as a key metabolite and signalling molecule in mammals, but the methods to investigate it in vivo have been limited. Here, authors report a pair of improved biosensors—one green and one red—for visualizing l-lactate both inside and outside of cells.
- Yusuke Nasu
- , Abhi Aggarwal
- & Robert E. Campbell
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Article
| Open AccessDe novo design of knotted tandem repeat proteins
This study reports the successful de novo design of a trefoil knotted protein fold for which the crystal structure agrees closely with the intended trefoil knot topology.
- Lindsey A. Doyle
- , Brittany Takushi
- & Philip Bradley
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Article
| Open AccessRepurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids
Biosensors have a wide number of potential applications, but rapidly constructing genetically encoded biosensors remains challenging. Here, authors report a method for rapidly converting ANL superfamily enzymes into biosensors for organic acids, based on their conformational changes upon binding.
- Jin Wang
- , Ning Xue
- & Meng Wang
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| Open AccessReversible photoregulation of cell-cell adhesions with opto-E-cadherin
Tools for high spatiotemporal control of cell-cell adhesions are lacking. Here, authors propose an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light.
- Brice Nzigou Mombo
- , Brent M. Bijonowski
- & Seraphine V. Wegner
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| Open AccessFluorescent sensors for imaging of interstitial calcium
The study of interstitial calcium remains challenging due to scarce methodology. Here, authors present ultra-low affinity genetically encoded fluorescent biosensors for monitoring calcium in intercellular spaces in living tissues.
- Ariel A. Valiente-Gabioud
- , Inés Garteizgogeascoa Suñer
- & Oliver Griesbeck
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Article
| Open AccessNanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera
Most current anti-coronavirus nanoparticle vaccines target epitopes within the RBD. Here, the authors developed nanoparticles displaying an array of spike fusion proteins derived from various coronaviruses and show that immunizing mice with these vaccines elicits broad and potent cross-reactive antibodies.
- Geoffrey B. Hutchinson
- , Olubukola M. Abiona
- & Kizzmekia S. Corbett-Helaire
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| Open AccessComputational remodeling of an enzyme conformational landscape for altered substrate selectivity
The ability to rationally remodel enzyme conformational landscapes to modify catalytic properties is limited. Here, the authors, using a computational procedure, redesign the conformational landscape of an aminotransferase to stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate.
- Antony D. St-Jacques
- , Joshua M. Rodriguez
- & Roberto A. Chica
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| Open AccessSingle-chain dimers from de novo immunoglobulins as robust scaffolds for multiple binding loops
Here the authors describe principles for de novo designing single-chain immunoglobulin dimers with interfaces diverging from those seen in antibodies, showing enhanced stability and both robustness and modularity for harboring multiple functional loops.
- Jorge Roel-Touris
- , Marta Nadal
- & Enrique Marcos
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| Open AccessDe novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8
Roy et al. describe a generalized method for computationally designing miniproteins selective for a single integrin heterodimer and conformational state. The designed αvβ6 inhibitor remains monomeric and maintains biological activity following aerosolization and shows excellent efficacy in bleomycin induced lung fibrosis.
- Anindya Roy
- , Lei Shi
- & David Baker
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| Open AccessStructure-based design of a strain transcending AMA1-RON2L malaria vaccine
Here the authors use structure-based design to engineer a single component immunogen that mimics the malaria parasite AMA1-RON2 complex required for invasion of host cells, and show that it elicits a potent strain-transcending antibody response in rats.
- Palak N. Patel
- , Thayne H. Dickey
- & Niraj H. Tolia
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Article
| Open AccessCD98hc is a target for brain delivery of biotherapeutics
New delivery platforms are needed to allow broader application of biotherapeutics for CNS diseases. Here, the authors show enhanced CNS delivery with a transport vehicle engineered to bind CD98hc, a highly expressed target at the blood-brain barrier.
- Kylie S. Chew
- , Robert C. Wells
- & Mihalis S. Kariolis
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| Open AccessProgramming conformational cooperativity to regulate allosteric protein-oligonucleotide signal transduction
Conformational cooperativity is a universal molecular effect mechanism and plays a critical role in signalling pathways. Here the authors present a programmable conformational cooperativity strategy to construct the oligo-protein signal transduction platform for logic operations and gene regulations which can be cooperatively regulated by conformational signals.
- Yuan Liang
- , Yunkai Qie
- & Cheng Zhang
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| Open AccessBiosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries
Engineering polyketide synthases can be challenging due to the absence of efficient high-throughput methods. Here, the authors used a solubility biosensor to identify stable variants from libraries of modified polyketide synthases.
- Elias Englund
- , Matthias Schmidt
- & Jay D. Keasling
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| Open AccessDiscovery and rational engineering of PET hydrolase with both mesophilic and thermophilic PET hydrolase properties
Extensive research efforts have been directed towards the development of PET hydrolases with improved activity, but template enzymes used are limited. Here, the authors report a PET hydrolase from Cryptosporangium aurantiacum (CaPETase) that exhibits high thermostability and PET degradation activity at ambient temperatures and determine its crystal structure.
- Hwaseok Hong
- , Dongwoo Ki
- & Kyung-Jin Kim
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Article
| Open AccessLiver lipophagy ameliorates nonalcoholic steatohepatitis through extracellular lipid secretion
Nonalcoholic steatohepatitis (NASH) starts with lipid droplet accumulation in the liver that eventually causes inflammation and fibrosis. Here, authors use lipophagy activators to limit the accumulation of lipids in the liver and show that this can prevent disease progression in a mouse model of nonalcoholic steatohepatitis.
- Yoshito Minami
- , Atsushi Hoshino
- & Satoaki Matoba
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Article
| Open AccessAn unnatural enzyme with endonuclease activity towards small non-coding RNAs
Endonucleases play crucial roles in various biological processes but endonucleases that target small non-coding RNAs have not been reported. Here, the authors combined the metal binding non-canonical amino acid BpyAla and a high affinity binder to engineer a catalyst that degrades small non-coding RNAs.
- Noreen Ahmed
- , Nadine Ahmed
- & John Paul Pezacki
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Article
| Open AccessIsolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli
Discovery of full-length antibodies can be slow and labor intensive. Here, the authors describe a robust genetic assay for facile isolation of IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered bacteria.
- Michael-Paul Robinson
- , Jinjoo Jung
- & Matthew P. DeLisa
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Article
| Open AccessA single-domain green fluorescent protein catenane
Natural proteins exhibit rich structural diversity based on the folds of an invariably linear chain. Here the authors design a single-domain GFP catenane as the counterpart of conventional linear GFP with enhanced thermal resilience and to provide a robust scaffold for making fusion protein catenanes.
- Zhiyu Qu
- , Jing Fang
- & Wen-Bin Zhang
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Article
| Open AccessAtomic model for core modifying region of human fatty acid synthase in complex with Denifanstat
Here, the authors develop a protein engineering method that enables high-resolution structural biology study of human fatty acid synthase. Using this technique, they uncover unique structural features of the enzyme and the mechanism of its inhibition by an anticancer drug Denifanstat.
- S. M. Naimul Hasan
- , Jennifer W. Lou
- & Mohammad T. Mazhab-Jafari
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Article
| Open AccessMachine learning optimization of candidate antibody yields highly diverse sub-nanomolar affinity antibody libraries
Therapeutic antibody discovery is time and cost-intensive. Here, the authors develop a machine learning-driven method enabling accelerated design of large and diverse single-chain variable fragments with high binding efficiency, especially at high levels of diversity.
- Lin Li
- , Esther Gupta
- & Matthew E. Walsh
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Article
| Open AccessDesigned active-site library reveals thousands of functional GFP variants
Mutations in a protein active site can alter function in useful ways, but the active site is sensitive to changes. Here the authors present a general strategy to design combinatorial mutation libraries. Applied to GFP, the authors isolate thousands of fluorescent designs that exhibit large and useful changes in spectral properties.
- Jonathan Yaacov Weinstein
- , Carlos Martí-Gómez
- & Sarel J. Fleishman
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Article
| Open AccessComputational design of dynamic receptor—peptide signaling complexes applied to chemotaxis
Engineering protein biosensors that respond to biomolecules by triggering cellular responses has largely relied on binding rigid molecules. Here, the authors develop a computational strategy for designing signaling complexes between conformationally dynamic proteins and peptides.
- Robert E. Jefferson
- , Aurélien Oggier
- & Patrick Barth
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Article
| Open AccessMetal3D: a general deep learning framework for accurate metal ion location prediction in proteins
Zinc is an essential metal for many proteins. Here, the authors propose a model based on 3D convolutional networks to predict the location of zinc in experimental and computationally predicted structures within a framework readily extensible to other metals.
- Simon L. Dürr
- , Andrea Levy
- & Ursula Rothlisberger
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Article
| Open AccessImproving de novo protein binder design with deep learning
Recently, a pipeline for the design of protein-binding proteins using only the structure of the target protein was reported. Here, the authors report that the incorporation of deep learning methods into the original pipeline increases experimental success rate by ten-fold.
- Nathaniel R. Bennett
- , Brian Coventry
- & David Baker