Protein design

  • Article
    | Open Access

    High-affinity antibodies are often identified through directed evolution but deep leaning methods hold great promise. Here the authors report RESP, a pipeline for efficient identification of high affinity antibodies, and apply this to the PD-L1 antibody Atezolizumab.

    • Jonathan Parkinson
    • , Ryan Hard
    •  & Wei Wang
  • Article
    | Open Access

    In this work the authors investigate the structure-sequence dependance. The ability to design and characterize proteins at interfaces between three common folds suggests that fold switching is an intrinsic feature of protein folding language and likely important in the evolution of protein structure and function.

    • Biao Ruan
    • , Yanan He
    •  & Philip N. Bryan
  • Article
    | Open Access

    Differential sensing aims to mimic senses such as taste and smell through the use of synthetic receptors. Here, the authors show that arrays of de novo designed peptide assemblies can be used as sensor components to distinguish various analytes and complex mixtures.

    • William M. Dawson
    • , Kathryn L. Shelley
    •  & Derek N. Woolfson
  • Article
    | Open Access

    Design of recombinases with new target sites is usually achieved through cycles of directed molecular evolution. Here the authors report Recombinase Generator, RecGen, an algorithm for generation of designer-recombinases; they perform experimental validation to show that this can predict recombinase sequences.

    • Lukas Theo Schmitt
    • , Maciej Paszkowski-Rogacz
    •  & Frank Buchholz
  • Article
    | Open Access

    Chiral heterocyclic compounds are privileged structures in medicinal chemistry. Here, the authors report an in silico strategy for the enzymatic synthesis of pharmaceutically significant chiral N- and O-heterocycles via Baldwin cyclization of hydroxy- and amino-substituted epoxides and oxetanes using epoxide hydrolase mutants.

    • Jun-Kuan Li
    • , Ge Qu
    •  & Zhoutong Sun
  • Article
    | Open Access

    Engineering enzymes to accept noncanonical cofactor biomimetics is difficult. Here, the authors establish a self-sufficient growth selection method and demonstrate its application in engineering the Lactobacillus pentosus NADH oxidase to efficiently recycle reduced nicotinamide mononucleotide (NMNH).

    • Edward King
    • , Sarah Maxel
    •  & Han Li
  • Article
    | Open Access

    In this work the authors provide a computational workflow for the parallel, from scratch, design of proteins to rapidly explore the shape diversity of protein folds.

    • Thomas W. Linsky
    • , Kyle Noble
    •  & Eva-Maria Strauch
  • Article
    | Open Access

    Molecular level control is required to capture the folding and supramolecular assembly of collagen in mimetic materials. Here, the authors report on the creation of a synthetic collagen which assembles into banded fibers, recaptures structural properties of natural collagen and which can act as a testbed for design and experimentation

    • Jinyuan Hu
    • , Junhui Li
    •  & Fei Xu
  • Article
    | Open Access

    The immunoglobulin domain framework of antibodies has been a long standing design challenge. Here, the authors describe design rules for tailoring these domains and show they can be accurately designed, de novo, with high stability and the ability to scaffold functional loops.

    • Tamuka M. Chidyausiku
    • , Soraia R. Mendes
    •  & Enrique Marcos
  • Article
    | Open Access

    Conditional Degron Tags are a valuable tool to validate and study novel therapeutic targets. Here, the authors compared 5 orthogonal tags across 16 unique proteins and provide a panel of vectors for users to systematically screen the tags with their own protein of interest.

    • Daniel P. Bondeson
    • , Zachary Mullin-Bernstein
    •  & Alessandra Ianari
  • Article
    | Open Access

    Efficient engineering of enzymes for expanded substrate scope is currently challenging. Here, the authors develop simple principles of how to design and interpret Substrate Multiplexed Screening assays to guide protein engineering to enable activity improvements with simultaneously with multiple substrates.

    • Allwin D. McDonald
    • , Peyton M. Higgins
    •  & Andrew R. Buller
  • Article
    | Open Access

    Arising through multiple binding elements, multivalency can specify the avidity, duration, cooperativity, and selectivity of biomolecular interactions, but quantitative prediction and design of these properties has remained challenging. Here the authors enable facile analysis and engineering of multivalent binding by developing MVsim, a simulator that incorporates biochemical and biophysical parameters of interacting molecules and is accessible through a graphical user interface.

    • Bence Bruncsics
    • , Wesley J. Errington
    •  & Casim A. Sarkar
  • Article
    | Open Access

    De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build enzymes for efficient energy conversion. Here, the authors develop an artificial photosynthetic reaction center that functions without the need for sacrificial electron donors or acceptors.

    • Nathan M. Ennist
    • , Zhenyu Zhao
    •  & Christopher C. Moser
  • Article
    | Open Access

    Approaches have been devised to increase the discovery rate of intrabodies but often these yield results that aren’t functional in cells. Here the authors engineer and optimise an autonomous and disulphide-free human VH domain for intracellular expression, and they identify several VH domain binders against eIF4E.

    • Yuri Frosi
    • , Yen-Chu Lin
    •  & Christopher J. Brown
  • Article
    | Open Access

    Bacteroides species are prominent members of the human gut microbiota. Here the authors develop a platform technology (consortium transcriptional programming) in five human donor Bacteroides chassis cells—enabling complex decision-making within said community, which can be used for the development of living therapeutics.

    • Brian D. Huang
    • , Thomas M. Groseclose
    •  & Corey J. Wilson
  • Article
    | Open Access

    Optimising antibody properties such as affinity can be detrimental to other key properties. Here the authors use machine learning to simplify the identification of antibodies with co-optimal levels of affinity and specificity for a clinical-stage antibody that displays high levels of on- and off-target binding.

    • Emily K. Makowski
    • , Patrick C. Kinnunen
    •  & Peter M. Tessier
  • Article
    | Open Access

    The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. Here, the authors use computational design to discover Cas9 enzymes with increased activity.

    • Pascal D. Vos
    • , Giulia Rossetti
    •  & Oliver Rackham
  • Article
    | Open Access

    Fluorescent biosensors are important tools for studying cellular metabolism, but development and optimization are challenging. Koveal et al. present a high-throughput multiparameter screen for sensor performance, and used it to generate LiLac, a high-performance, quantitative lactate sensor.

    • Dorothy Koveal
    • , Paul C. Rosen
    •  & Gary Yellen
  • Article
    | Open Access

    Dynamic control over protein function is a central challenge in synthetic biology. Here the authors present an integrated computational and experimental workflow for engineering reversible protein switches; metal-chelating unnatural amino acids genetically encoded into two conformationally dynamic enzymes to yield robust switches.

    • Yasmine S. Zubi
    • , Kosuke Seki
    •  & Jared C. Lewis
  • Article
    | Open Access

    Influenza virus neuraminidase (NA) is a drug target and a potential vaccine antigen. Here, the authors provide a detailed analysis of the conformational stability of NA, and show how expression and stability of recombinant NA antigens can be strengthened through structure-based design.

    • Daniel Ellis
    • , Julia Lederhofer
    •  & Masaru Kanekiyo
  • Article
    | Open Access

    Gq proteins are one of four major classes of G proteins; optogenetic receptors for selective and repetitive activation of Gq proteins with fast kinetics are lacking. Here the authors report UV light-dependent Gq signalling using human Neuropsin (hOPN5) and demonstrate its potential as an optogenetic tool.

    • Ahmed Wagdi
    • , Daniela Malan
    •  & Tobias Bruegmann
  • Article
    | Open Access

    Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Here the authors report de novo design and directed folding of peptide heterodimers crosslinked through multiple disulfide bonds, which can be explored as chemical tools for orthogonal labeling of proteins and preparing protein hybrids.

    • Sicong Yao
    • , Adam Moyer
    •  & Chuanliu Wu
  • Article
    | Open Access

    Rational protein design to achieve a given protein backbone conformation is needed to engineer specific functions. Here Anand et al. describe a machine learning method using a learned neural network potential for fixed-backbone protein design.

    • Namrata Anand
    • , Raphael Eguchi
    •  & Po-Ssu Huang
  • Article
    | Open Access

    The late-stage functionalization of unactivated carbon–hydrogen bonds is a difficult but important task, which has been met with promising but limited success through synthetic organic chemistry. Here the authors use machine learning to engineer WelO5* halogenase variants, which led to regioselective chlorination of inert C–H bonds on a representative polyketide that is a non-natural substrate for the enzyme.

    • Johannes Büchler
    • , Sumire Honda Malca
    •  & Rebecca Buller
  • Article
    | Open Access

    SxtT and GxtA are Rieske oxygenases that are involved in paralytic shellfish toxin biosynthesis and catalyze monohydroxylation reactions at different positions on the toxin scaffold. Here, the authors present crystal structures of SxtT and GxtA with the native substrates β-saxitoxinol and saxitoxin as well as a Xenon-pressurized structure of GxtA, which reveal a substrate access tunnel to the active site. Through structure-based mutagenesis studies the authors identify six residues in three different protein regions that determine the substrate specificity and site selectivity of SxtT and GxtA. These findings will aid the rational engineering of other Rieske oxygenases.

    • Jianxin Liu
    • , Jiayi Tian
    •  & Jennifer Bridwell-Rabb
  • Article
    | Open Access

    Here, we present TP-DB; a pattern-based search engine based on 1.67 million helices from the Protein Database (PDB). We demonstrate the utility of TP-DB in identifying microbe-specific antigens, as well as the design of antimicrobial peptides and Protein-protein interaction blockers.

    • Cheng-Yu Tsai
    • , Emmanuel Oluwatobi Salawu
    •  & Lee-Wei Yang
  • Article
    | Open Access

    Shark antibodies (Variable New Antigen Receptors, VNARs) are the smallest naturally occurring antibody fragments. Here, the authors screen a VNAR phage display library against the SARS-CoV2 receptor binding domain (RBD) and identify VNARs that neutralize the SARSCoV-2 virus and discuss their mechanisms of viral neutralization.

    • Obinna C. Ubah
    • , Eric W. Lake
    •  & Caroline J. Barelle
  • Article
    | Open Access

    Here, the authors perform a large-scale, high-throughput biochemical assay to determine the compatibility of over 300,000 domain recombination variants of the inward rectifier K+ channel Kir2.1. They derive rules for designing domain insertion variants that fold and traffic to the cell surface and conclude that the insertion of domains at protein termini is evolutionary favoured.

    • Willow Coyote-Maestas
    • , David Nedrud
    •  & Daniel Schmidt
  • Article
    | Open Access

    l-lactate is an important intercellular energy currency. Here the authors report a genetically encoded biosensor eLACCO1.1 to monitor extracellular l-lactate; they use eLACCO1.1 to image extracellular l-lactate in cultured mammalian cells and brain tissue.

    • Yusuke Nasu
    • , Ciaran Murphy-Royal
    •  & Robert E. Campbell
  • Article
    | Open Access

    Facilitated dissociation is a mechanism where antibody-mediated disruption of high-affinity complexes can enhance the therapeutic effects of a drug. Here the authors present a yeast display approach to select and engineer omalizumab variants that dissociate receptor-bound IgE to accelerate its inhibition of the allergic response.

    • Luke F. Pennington
    • , Pascal Gasser
    •  & Theodore S. Jardetzky
  • Article
    | Open Access

    Rational design of enzymes with new or improved properties is rarely straightforward, and artificial selection pressure approaches that link an improvement in the target to cell growth are an alternative. Here, the authors show that diverse enzymes sharing the ubiquitous cofactor NAD(P)+ can substitute for defective NAD+ regeneration, representing a very broadly-applicable artificial selection.

    • Lara Sellés Vidal
    • , James W. Murray
    •  & John T. Heap
  • Article
    | Open Access

    Certain bovine antibodies have ultra-long long complementarity-determining regions (CDRs) that contain a knob for antigen interaction, which is connected to the antibody through a stalk. Here, the authors combine biophysical experiments and MD simulations and show that the stalk length is critical for the folding and stability of these antibodies. The authors also demonstrate that ultra-long bovine CDRs can be grafted into human antibodies, and furthermore show that de novo designed mini-domains that bind to the SARS-CoV-2 spike protein with high affinity can be integrated as a knob in ultra-long CDRs in bovine and human antibodies, which neutralize SARS-CoV-2.

    • Hristo L. Svilenov
    • , Julia Sacherl
    •  & Johannes Buchner
  • Article
    | Open Access

    The directed evolution of antibodies yields important tools for research and therapy. Here the authors develop a periplasmic phage-assisted continuous evolution platform for improvement of protein-protein interactions in the disulfidecompatible E. coli periplasm.

    • Mary S. Morrison
    • , Tina Wang
    •  & David R. Liu
  • Article
    | Open Access

    Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions into OFF- and ON-switches active in cells.

    • Sailan Shui
    • , Pablo Gainza
    •  & Bruno E. Correia
  • 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