Protein design

  • Article
    | Open Access

    Faster, higher throughput antibody engineering methods are needed. Here the authors present CeVICA, a cell-free nanobody engineering platform using ribosome display and computational clustering analysis for in vitro selection; they use this to develop nanobodies against the RBD of SARS-CoV-2 spike protein.

    • Xun Chen
    • , Matteo Gentili
    •  & Aviv Regev
  • Article
    | Open Access

    Circularised nanodiscs (cNDs) are able to stabilise large lipid bilayer patches and are used for structural and functional studies. Current techniques to build cNDs have numerous steps and low yields; here the authors report a single step construction method using the SpyCatcher-SpyTag system.

    • Shanwen Zhang
    • , Qian Ren
    •  & Huan Bao
  • Article
    | Open Access

    Current near-IR optogenetic systems to regulate transcription consist of a number of large protein components. Here the authors report a smaller single-component near-IR system, iLight, developed from a bacterial phytochrome that they use to control gene transcription in bacterial and mammalian cells.

    • Andrii A. Kaberniuk
    • , Mikhail Baloban
    •  & Vladislav V. Verkhusha
  • Article
    | Open Access

    α-Synuclein (αS) aggregation is a driver of several neurodegenerative disorders. Here, the authors identify a class of peptides that bind toxic αS oligomers and amyloid fibrils but not monomeric functional protein, and prevent further αS aggregation and associated cell damage.

    • Jaime Santos
    • , Pablo Gracia
    •  & Salvador Ventura
  • Article
    | Open Access

    Directed evolution commonly relies on point mutations but InDels frequently occur in evolution. Here the authors report a protein-engineering framework based on InDel mutagenesis and fragment transplantation resulting in greater catalysis and longer glow-type bioluminescence of the ancestral luciferase.

    • Andrea Schenkmayerova
    • , Gaspar P. Pinto
    •  & Jiri Damborsky
  • Article
    | Open Access

    Self-assembling peptides (SAPs) can be used to build biomaterials, but genetically encoded SAPs have rarely been used as building blocks in cells. Here, the authors design a SAP that can be genetically fused to target proteins to induce their intracellular clustering and modulate their signaling functions.

    • Takayuki Miki
    • , Taichi Nakai
    •  & Hisakazu Mihara
  • Article
    | Open Access

    Cyclic peptides are of particular interest due to their pharmacological properties, but their design for binding to a target protein is challenging. Here, the authors present a computational “anchor extension” methodology for de novo design of cyclic peptides that bind to the target protein with high affinity, and validate the approach by developing cyclic peptides that inhibit histone deacetylases 2 and 6.

    • Parisa Hosseinzadeh
    • , Paris R. Watson
    •  & David Baker
  • Article
    | Open Access

    The ability to design functional sequences is central to protein engineering and biotherapeutics. Here the authors introduce a deep generative alignment-free model for sequence design applied to highly variable regions and design and test a diverse nanobody library with improved properties for selection experiments.

    • Jung-Eun Shin
    • , Adam J. Riesselman
    •  & Debora S. Marks
  • Article
    | Open Access

    De novo design of self-assembling protein nanostructures and materials is of significant interest, however design of complex, multi-component assemblies is challenging. Here, the authors present a stepwise hierarchical approach to build such assemblies using helical repeat and helical bundle proteins as building blocks, and provide an in-depth structural characterization of the resulting assemblies.

    • Yang Hsia
    • , Rubul Mout
    •  & David Baker
  • Article
    | Open Access

    Buried charged networks in proteins are often important for their biological functionality and are believed to destabilise the protein fold. Here, the authors combine computational design, MD simulations, biophysical experiments, NMR and X-ray crystallography to design and characterise artificial 4α-helical proteins with buried charged elements. They analyse their conformational landscapes and observe that the ion-pairs are stabilised by amphiphilic residues that electrostatically shield the charged motif, which increases structural stability.

    • Mona Baumgart
    • , Michael Röpke
    •  & Ville R. I. Kaila
  • Article
    | Open Access

    TEM-1 β-lactamase evolved from ancestral enzymes that degraded a variety of β-lactam antibiotics with moderate efficiency and degrades β-lactam antibiotics with a strong preference for penicillins. Here authors developed a computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism and show a putative Precambrian β-lactamase that mimics the modern TEM-1 β-lactamase with only 21 amino acid replacements.

    • Tushar Modi
    • , Valeria A. Risso
    •  & S. Banu Ozkan
  • Article
    | Open Access

    So far most of the de novo designed proteins are for single states only. Here, the authors present the de novo design and crystal structure determination of a coiled-coil peptide that assembles into multiple, distinct conformational states under the same conditions and further characterise its properties with biophysical experiments, NMR and MD simulations.

    • William M. Dawson
    • , Eric J. M. Lang
    •  & Derek N. Woolfson
  • Article
    | Open Access

    RaPID (Random non-standard Peptides Integrated Discovery) enables discovery of small macrocyclic peptides binding desired targets. Here, the authors propose lasso-grafting: the RaPID-derived peptides are implanted onto diverse proteins and maintain both the binding properties of the cyclic peptide and the host protein function.

    • Emiko Mihara
    • , Satoshi Watanabe
    •  & Junichi Takagi
  • Article
    | Open Access

    The ability to dynamically control protein-protein interactions and localization of proteins is critical in synthetic biological systems. Here the authors develop a peptide-based molecular switch that regulates dimer formation and lipid membrane targeting via reversible phosphorylation.

    • Leon Harrington
    • , Jordan M. Fletcher
    •  & Petra Schwille
  • Article
    | Open Access

    Active-site loops are important for catalytic properties of enzymes, but challenging to engineer due to their high flexibility and diversity. Here, the authors identify and engineer hot-spots in the loops of cumene dioxygenase, obtain variants with changed activity, regio- and enantioselectivity, and present a Linker In Loop Insertion approach for loop modification.

    • Peter M. Heinemann
    • , Daniel Armbruster
    •  & Bernhard Hauer
  • Article
    | Open Access

    Coiled-coil protein origami is a strategy for the de novo design of polypeptide nanostructures based on coiled-coil dimer forming peptides, where a single chain protein folds into a polyhedral cage. Here, the authors design a single-chain triangular bipyramid and also demonstrate that the bipyramid can be self-assembled as a heterodimeric complex, comprising pre-defined subunits.

    • Fabio Lapenta
    • , Jana Aupič
    •  & Roman Jerala
  • Article
    | Open Access

    Coiled-coil protein origami (CCPO) is a strategy for the design of polyhedral cage-shaped protein folds based on coiled-coil (CC) dimer-forming peptides. Here, the authors show that CCPO proteins fold in a multistep process governed by the spatial distance between CC modules in the primary sequence and subsequent folding intermediates, which enables the use of identical CC modules for the CCPO tetrahedron design.

    • Jana Aupič
    • , Žiga Strmšek
    •  & Roman Jerala
  • Article
    | Open Access

    Fluorescent protein reporters based on GFP exist, but have intrinsic disadvantages. Here the authors incorporate pH, Ca2+ and protein–protein interaction sensing modalities into de novo designed mini-fluorescence-activating proteins (mFAPs), with increased photostability and smaller size, which bind a range of DFHBI chromophore variants.

    • Jason C. Klima
    • , Lindsey A. Doyle
    •  & David Baker
  • Article
    | Open Access

    Covalent conjugation of endogenous protein complexes offers many opportunities for fundamental and clinical research. Based on a bacterial protein domain that forms a reactive anhydride in the presence of Ca2+, the authors here develop a system that enables the covalent capture of endogenous binding partners.

    • Arne H. A. Scheu
    • , Sheryl Y. T. Lim
    •  & Mark Howarth
  • Article
    | Open Access

    Family 1 glycosidases (GH1) are present in the three domains of life and share classical TIM-barrel fold. Structural and biochemical analyses of a resurrected ancestral GH1 enzyme reveal heme binding, not known in its modern descendants. Heme rigidifies the TIM-barrel and allosterically enhances catalysis.

    • Gloria Gamiz-Arco
    • , Luis I. Gutierrez-Rus
    •  & Jose M. Sanchez-Ruiz
  • Article
    | Open Access

    Existing optogenetic methods to induce calcium mobilisation lack selectivity and specificity. Here, the authors design and engineer a single-component light-operated calcium channel to provide optical control over calcium signals and calcium-dependent physiological responses: LOCa.

    • Lian He
    • , Liuqing Wang
    •  & Yubin Zhou
  • Article
    | Open Access

    Protein cages are used for encapsulation of macromolecules such as proteins and nucleic acids. Here, the authors report a strategy to encapsulate poorly soluble small molecules within porous protein cages through capsid-templated micelle formation and show that the resulting lipoprotein-like cages enhance the cellular delivery of these molecules.

    • Thomas G. W. Edwardson
    • , Stephan Tetter
    •  & Donald Hilvert
  • Article
    | Open Access

    ADP-ribose binding macro domains facilitate the enrichment and detection of cellular ADP-ribosylation. Here, the authors generate an engineered macro domain with increased ADP-ribose affinity, improving the identification of ADP-ribosylated proteins by proteomics, western blot and immunofluorescence.

    • Kathrin Nowak
    • , Florian Rosenthal
    •  & Michael O. Hottiger
  • Article
    | Open Access

    Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunomodulatory receptors expressed on cells of the hematopoietic lineage. Here the authors demonstrate an approach for the identification of the glycan ligands of Siglecs, which is also applicable to other families of glycan-binding proteins.

    • Emily Rodrigues
    • , Jaesoo Jung
    •  & Matthew S. Macauley
  • Article
    | Open Access

    Kemp eliminases are artificial enzymes that catalyze the concerted deprotonation and ring-opening of benzisoxazoles. Here, the authors use room-temperature X-ray crystallography to investigate changes to the conformational ensemble of the Kemp eliminase HG3 along a directed evolutionary trajectory, and develop an experimentally guided, ensemble-based computational enzyme design procedure.

    • Aron Broom
    • , Rojo V. Rakotoharisoa
    •  & Roberto A. Chica
  • Article
    | Open Access

    Mechanically stable specific heterodimerization formed with reversible bonds are used as a molecular anchorage in single-molecule force spectroscopy studies with unique mechanical properties. Here authors develop a variety of heterodimerization molecular systems with a range of mechanical stability from a set of recently engineered helix-heterotetramers.

    • Miao Yu
    • , Zhihai Zhao
    •  & Jie Yan
  • Article
    | Open Access

    Backbone extended monomers are poorly compatible with the natural ribosomes, impeding their polymerization into polypeptides. Here the authors design non-canonical amino acid analogs with cyclic structures or extended carbon chains and used an engineered ribosome to improve tRNA-charging and incorporation into peptides.

    • Joongoo Lee
    • , Kevin J. Schwarz
    •  & Michael C. Jewett
  • Article
    | Open Access

    The ability to reversibly control monobody binding affinity would find use in biotechnology and research applications. Here the authors fuse the light-sensitive AsLOV2 domain to a monobody against the Abl SH2 domain to obtain a light dependent monobody and apply it in vitro and in mammalian cells.

    • César Carrasco-López
    • , Evan M. Zhao
    •  & José L. Avalos
  • Article
    | Open Access

    Genetically encoded voltage indicators (GEVIs) allow visualisation of fast action potentials in neurons but most are bright at rest and dimmer during an action potential. Here, the authors engineer electrochromic FRET GEVIs with fast, bright and positive-going fluorescence signals for in vivo imaging.

    • Ahmed S. Abdelfattah
    • , Rosario Valenti
    •  & Eric R. Schreiter
  • Article
    | Open Access

    Glutaredoxins are a family of essential enzymes divided into two major classes with either a CGFS or a CxxC active site, of which only the latter exhibits oxidoreductase activity. Here the authors address the structural basis for the functional difference between the two classes of glutaredoxins.

    • Daniel Trnka
    • , Anna D. Engelke
    •  & Christopher Horst Lillig
  • Article
    | Open Access

    Malate dehydrogenase (MDH) is one of the key enzymes for succinic acid (SA) bioproduction. Here, the authors report biochemical and structural analyses of various MDHs to reveal amino acids influencing the specific activity and susceptibility to substrate inhibition, and achieve industrial-level SA production.

    • Jung Ho Ahn
    • , Hogyun Seo
    •  & Sang Yup Lee
  • Article
    | Open Access

    The spectra of light used by photosynthetic organisms are determined by their pigmentation colour palettes. Here Liu et al. show that a genetically-encoded chimera of light-harvesting proteins from plants and reaction centres from purple bacteria allows for polychromatic solar energy harvesting.

    • Juntai Liu
    • , Vincent M. Friebe
    •  & Michael R. Jones
  • Article
    | Open Access

    Major Histocompatibility Complex (MHC) class I molecules present tightly binding peptides on the cell surface for recognition by cytotoxic T cells. Here, the authors present the crystal structures of a disulfide-stabilized human MHC class I molecule in the peptide-free state and bound with dipeptides, and find that peptide binding is accompanied by concerted conformational switches of the amino acid side chains in the binding pockets.

    • Raghavendra Anjanappa
    • , Maria Garcia-Alai
    •  & Rob Meijers
  • Article
    | Open Access

    CRISPR homing gene drives are highly invasive and can fail due to the rapid evolution of resistance. Here the authors present TARE drive, inspired by naturally occurring selfish genetic elements, which is less vulnerable to resistance and can potentially be confined to a target population.

    • Jackson Champer
    • , Esther Lee
    •  & Philipp W. Messer
  • Article
    | Open Access

    Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Here the authors report 3-bromo-5-methylene pyrrolones as a simple, robust and versatile class of reagents for cysteine-specific protein modification.

    • Yingqian Zhang
    • , Chuanlong Zang
    •  & Chuanzheng Zhou
  • Article
    | Open Access

    Protein tyrosine phosphatases regulate many cellular processes but are difficult to study in their native context. Here the authors develop an approach for using light to control the activity of a disease-relevant phosphatase without interfering with its native cellular organization.

    • Akarawin Hongdusit
    • , Peter H. Zwart
    •  & Jerome M. Fox
  • Article
    | Open Access

    Antibodies expressed in the cytosol often form insoluble aggregates, which makes it hard to target intracellular proteins. Here the authors engineer an ultra-stable cytoplasmic antibody (STAND) with a low isoelectric point that can be used in vivo.

    • Hiroyuki Kabayama
    • , Makoto Takeuchi
    •  & Katsuhiko Mikoshiba
  • Article
    | Open Access

    Monomeric near-infrared (NIR) fluorescent proteins (FPs) from bacterial phytochromes bring potential advantages, but their brightness in cells is lower than dimeric NIR FPs. Here the authors develop enhanced monomeric NIR FPs enabling imaging across different scales without the trade-off between brightness and monomeric state.

    • Mikhail E. Matlashov
    • , Daria M. Shcherbakova
    •  & Vladislav V. Verkhusha