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Sequence-activity mapping via depletion reveals striking mutational tolerance and elucidates functional motifs in Tur1a antimicrobial peptide Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2024-03-12 Jonathan Collins, Benjamin J Hackel
Proline-rich antimicrobial peptides (PrAMPs) are attractive antibiotic candidates that target gram-negative bacteria ribosomes. We elucidated the sequence-function landscape of 43 000 variants of a recently discovered family member, Tur1a, using the validated SAMP-Dep platform that measures intracellular AMP potency in a high-throughput manner via self-depletion of the cellular host. The platform exhibited
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The shortest path method (SPM) webserver for computational enzyme design Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2024-03-03 Guillem Casadevall, Jordi Casadevall, Cristina Duran, Sílvia Osuna
SPMweb is the online webserver of the Shortest Path Map (SPM) tool for identifying the key conformationally-relevant positions of a given enzyme structure and dynamics. The server is built on top of the DynaComm.py code and enables the calculation and visualization of the SPM pathways. SPMweb is easy-to-use as it only requires three input files: the three-dimensional structure of the protein of interest
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Design of functional intrinsically disordered proteins Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2024-03-01 Ankush Garg, Nicolas S González-Foutel, Maciej B Gielnik, Magnus Kjaergaard
Many proteins do not fold into a fixed three-dimensional structure, but rather function in a highly disordered state. These intrinsically disordered proteins pose a unique challenge to protein engineering and design: How can proteins be designed de novo if not by tailoring their structure? Here, we will review the nascent field of design of intrinsically disordered proteins with focus on applications
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Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2024-02-01 Nandan Haloi, Shan Huang, Aaron L Nichols, Eve J Fine, Nicholas J Friesenhahn, Christopher B Marotta, Dennis A Dougherty, Erik Lindahl, Rebecca J Howard, Stephen L Mayo, Henry A Lester
We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0 vs [nicotine] (δ-slope, 2.7 μM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance
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Protein sequence design on given backbones with deep learning Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-12-29 Yufeng Liu, Haiyan Liu
Deep learning methods for protein sequence design focus on modeling and sampling the many- dimensional distribution of amino acid sequences conditioned on the backbone structure. To produce physically foldable sequences, inter-residue couplings need to be considered properly. These couplings are treated explicitly in iterative methods or autoregressive methods. Non-autoregressive models treating these
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Growing ecosystem of deep learning methods for modeling protein–protein interactions Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-12-15 Julia R Rogers, Gergö Nikolényi, Mohammed AlQuraishi
Numerous cellular functions rely on protein–protein interactions. Efforts to comprehensively characterize them remain challenged however by the diversity of molecular recognition mechanisms employed within the proteome. Deep learning has emerged as a promising approach for tackling this problem by exploiting both experimental data and basic biophysical knowledge about protein interactions. Here, we
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De novo Design of a Polycarbonate Hydrolase Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-12-01 Laura H Holst, Niklas G Madsen, Freja T Toftgård, Freja Rønne, Ioana-Malina Moise, Evamaria I Petersen, Peter Fojan
Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural $\alpha $/$\beta $-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the $\alpha $/$\beta $-fold. It is thus of interest to introduce catalytic activity toward
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abYpap: Improvements to the Prediction of Antibody V H/V L Packing Using Gradient Boosted Regression Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-11-28 Veronica A Boron, Andrew C R Martin
The Fv region of the antibody (comprising VH and VL domains) is the area responsible for target binding and thus the antibody’s specificity. The orientation, or packing, of these two domains relative to each other influences the topography of the Fv region, and therefore can influence the antibody’s binding affinity. We present abYpap, an improved method for predicting the packing angle between the
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A protein engineering approach towards understanding FKBP51 conformational dynamics and mechanisms of ligand binding Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-10-30 Jorge A Lerma Romero, Christian Meyners, Nicole Rupp, Felix Hausch, Harald Kolmar
Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51)
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Analysis of conformational stability of interacting residues in protein binding interfaces Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-10-27 Varun M Chauhan, Robert J Pantazes
After approximately 60 years of work, the protein folding problem has recently seen rapid advancement thanks to the inventions of AlphaFold and RoseTTAFold, which are machine-learning algorithms capable of reliably predicting protein structures from their sequences. A key component in their success was the inclusion of pairwise interaction information between residues. As research focus shifts towards
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Physics-based approach to extend a de novo TIM barrel with rationally designed helix–loop–helix motifs Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-09-13 Sina Kordes, Julian Beck, Sooruban Shanmugaratnam, Merle Flecks, Birte Höcker
Computational protein design promises the ability to build tailor-made proteins de novo. While a range of de novo proteins have been constructed so far, the majority of these designs have idealized topologies that lack larger cavities which are necessary for the incorporation of small molecule binding sites or enzymatic functions. One attractive target for enzyme design is the TIM-barrel fold, due
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An easy-to-use high-throughput selection system for the discovery of recombinant protein binders from alternative scaffold libraries Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-09-12 Marit Möller, Malin Jönsson, Magnus Lundqvist, Blenda Hedin, Louise Larsson, Emma Larsson, Johan Rockberg, Mathias Uhlén, Sarah Lindbo, Hanna Tegel, Sophia Hober
Selection by phage display is a popular and widely used technique for the discovery of recombinant protein binders from large protein libraries for therapeutic use. The protein library is displayed on the surface of bacteriophages which are amplified using bacteria, preferably Escherichia coli, to enrich binders in several selection rounds. Traditionally, the so-called panning procedure during which
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Engineering Candida boidinii formate dehydrogenase for activity with the non-canonical cofactor 3’-NADP(H) Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-08-30 Salomon Vainstein, Scott Banta
Oxidoreductases catalyze critical essential redox reactions, and many of these enzymes require a diffusible secondary substrate, or cofactor, for electron transport. Nicotinamide adenine dinucleotide NAD(H) and nicotinamide adenine dinucleotide phosphate (NADP(H)) differ by a 2′-phosphate group and their inability to cross-react allows these cofactor pools to be used by a number of cellular processes
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The variable conversion of neutralizing anti-SARS-CoV-2 single chain antibodies to IgG provides insight into RBD epitope accessibility Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-08-10 Matthew R Chang, Hanzhong Ke, Laura Losada Miguéns, Christian Coherd, Katrina Nguyen, Maliwan Kamkaew, Rebecca Johnson, Nadia Storm, Anna Honko, Quan Zhu, Anthony Griffiths, Wayne A Marasco
Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe respiratory syncytial virus infections in neonatal
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Contributions from ClpS surface residues in modulating N-terminal peptide binding and their implications for NAAB development Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-07-27 Nicholas W Callahan, William B Siegall, Christina Bergonzo, John P Marino, Zvi Kelman
Numerous technologies are currently in development for use in next-generation protein sequencing platforms. A notable published approach employs fluorescently-tagged binding proteins to identity the N-terminus of immobilized peptides, in-between rounds of digestion. This approach makes use of N-terminal amino acid binder (NAAB) proteins, which would identify amino acids by chemical and shape complementarity
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Enhancing the activity of a monomeric alcohol dehydrogenase for site-specific applications by site-directed mutagenesis Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-06-12 Arabella Essert, Kathrin Castiglione
Gene fusion or co-immobilization are key tools to optimize enzymatic reaction cascades by modulating catalytic features, stability and applicability. Achieving a defined spatial organization between biocatalysts by site-specific applications is complicated by the involvement of oligomeric enzymes. It can lead to activity losses due to disturbances of the quaternary structures and difficulties in stoichiometric
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Yeast biopanning against site-specific phosphorylations in tau Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-06-09 Monika Arbaciauskaite, Azady Pirhanov, Erik Ammermann, Yu Lei, Yong Ku Cho
Antibodies that bind to protein phosphorylation sites are a critical tool for detecting the post-translational modification. The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer’s Disease (AD) as well as other neurodegenerative diseases. However, the need for reliable phospho-site specific
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Enzyme design pioneer Steve Mayo: I was trying to capture the fundamental physics of the problem as a way to elucidate mechanisms. Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-01-21 Roberto A Chica,Brett M Garabedian
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Non-canonical amino acids as a tool for the thermal stabilization of enzymes Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-03-10 Tim Lugtenburg, Alejandro Gran-Scheuch, Ivana Drienovská
Biocatalysis has become a powerful alternative for green chemistry. Expanding the range of amino acids used in protein biosynthesis can improve industrially appealing properties such as enantioselectivity, activity and stability. This review will specifically delve into the thermal stability improvements that non-canonical amino acids (ncAAs) can confer to enzymes. Methods to achieve this end, such
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Engineering cellulases for conversion of lignocellulosic biomass Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-03-09 Yogesh Babasaheb Chaudhari, Anikó Várnai, Morten Sørlie, Svein Jarle Horn, Vincent G H Eijsink
Lignocellulosic biomass is a renewable source of energy, chemicals, and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases (LPMOs) is a prerequisite for economically viable exploitation of this
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Engineering of a phosphotriesterase with improved stability and enhanced activity for detoxification of the pesticide metabolite malaoxon. Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-01-21 Laura Job,Anja Köhler,Mauricio Testanera,Benjamin Escher,Franz Worek,Arne Skerra
Organophosphorus (OP) pesticides are still widely applied but pose a severe toxicological threat if misused. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with
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Enzyme redesign and genetic code expansion. Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-01-21 Vaitea Opuu,Thomas Simonson
Enzyme design is an important application of computational protein design (CPD). It can benefit enormously from the additional chemistries provided by noncanonical amino acids (ncAAs). These can be incorporated into an 'expanded' genetic code, and introduced in vivo into target proteins. The key step for genetic code expansion is to engineer an aminoacyl-transfer RNA (tRNA) synthetase (aaRS) and an
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Benchmarking TriadAb using targets from the second antibody modeling assessment. Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-01-21 Frederick S Lee,Amos G Anderson,Barry D Olafson
Computational modeling and design of antibodies has become an integral part of today's research and development in antibody therapeutics. Here we describe the Triad Antibody Homology Modeling (TriadAb) package, a functionality of the Triad protein design platform that predicts the structure of any heavy and light chain sequences of an antibody Fv domain using template-based modeling. To gauge the performance
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Strategies for enriching and characterizing proteins with inhibitory properties on the yeast surface Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-01-17 Arlinda Rezhdo, Catherine T Lessard, Mariha Islam, James A Van Deventer
Display technologies are powerful tools for discovering binding proteins against a broad range of biological targets. However, it remains challenging to adapt display technologies for the discovery of proteins that inhibit the enzymatic activities of targets. Here, we investigate approaches for discovering and characterizing inhibitory antibodies in yeast display format using a well-defined series
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Effect of alanine versus serine at position 88 of human transthyretin mutants on the protein stability Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2023-01-02 Kyung-Hoon Lee, Krzysztof Kuczera
Human Transthyretin (TTR) is a homo-tetrameric plasma protein associated with a high percentage of β-sheet forming amyloid fibrils. It accumulates in tissues or extracellular matrices to cause amyloid diseases. Free energy simulations with thermodynamic integration based on all-atom molecular dynamics simulations have been carried out to analyze the effects of the His88 → Ala and Ser mutations on the
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Bispecific antibodies-effects of point mutations on CH3-CH3 interface stability. Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-02-17 Nancy D Pomarici,Monica L Fernández-Quintero,Patrick K Quoika,Franz Waibl,Alexander Bujotzek,Guy Georges,Klaus R Liedl
A new format of therapeutic proteins is bispecific antibodies, in which two different heavy chains heterodimerize to obtain two different binding sites. Therefore, it is crucial to understand and optimize the third constant domain (CH3-CH3) interface to favor heterodimerization over homodimerization, and to preserve the physicochemical properties, as thermal stability. Here, we use molecular dynamics
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Structure-based engineering of minimal Proline dehydrogenase domains for inhibitor discovery Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-11-30 Alexandra N Bogner, Juan Ji, John J Tanner
Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of L-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations
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Engineering the Enzyme Toolbox to Tailor Glycosylation in Small Molecule Natural Products and Protein Biologics Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-11-25 Sara Ouadhi, Dulce María Valdez López, F Ifthiha Mohideen, David H Kwan
Many glycosylated small molecule natural products and glycoprotein biologics are important in a broad range of therapeutic and industrial applications. The sugar moieties that decorate these compounds often show a profound impact on their biological functions, thus biocatalytic methods for controlling their glycosylation are valuable. Enzymes from nature are useful tools to tailor bioproduct glycosylation
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Integrating dynamics into enzyme engineering Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-11-21 Claudèle Lemay-St-Denis, Nicolas Doucet, Joelle N Pelletier
Enzyme engineering has become a widely adopted practice in research labs and industry. In parallel, the past decades have seen tremendous strides in characterizing the dynamics of proteins, using a growing array of methodologies. Importantly, links have been established between the dynamics of proteins and their function. Characterizing the dynamics of an enzyme prior to, and following, its engineering
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Engineering enzyme activity using an expanded amino acid alphabet Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-11-12 Zachary Birch-Price, Christopher J Taylor, Mary Ortmayer, Anthony P Green
Enzyme design and engineering strategies are typically constrained by the limited size of nature’s genetic alphabet, comprised of only twenty canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, and hundreds of structurally diverse amino
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A multivalent antibody assembled from different building blocks using tag/catcher systems: A case study Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-11-11 Christof Schindler, Christine Faust, Hanno Sjuts, Christian Lange, Jennifer Kühn, Werner Dittrich, Wulf Dirk Leuschner, Werner Schiebler, Joachim Hofmann, Ercole Rao, Thomas Langer
The field of therapeutic antibodies and especially bi- or multispecific antibodies is growing rapidly. Especially for treating cancers, multispecific antibodies are very promising as there are multiple pathways involved and multispecific antibodies offer the possibility to interfere at two or more sites. Besides being used as therapeutic, multispecific antibodies can be helpful tools in basic research
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Antibody-mediated delivery of CRISPR-Cas9 ribonucleoproteins in human cells Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-11-07 Stephanie Ubiparipovic, Daniel Christ, Romain Rouet
The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease or muscular dystrophy. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells and/or tissues. However, such methods are currently limited to direct organ delivery, preventing wide
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Reducing substrate inhibition of malate dehydrogenase from Geobacillus stearothermophilus by C-terminal truncation Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-10-08 Yuya Shimozawa, Hinano Matsuhisa, Tsutomu Nakamura, Tomoki Himiyama, Yoshiaki Nishiya
Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate to L-malate using NADH as a cofactor. Geobacillus stearothermophilus MDH (gs-MDH) is used as a diagnostic reagent; however, gs-MDH is robustly inhibited at high substrate concentrations, which limits its reaction rate. Here, we reduced substrate inhibition of gs-MDH by deleting its C-terminal residues. Computational analysis showed
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Data-Driven Enzyme Engineering to Identify Function-Enhancing Enzymes Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-10-06 Yaoyukun Jiang, Xinchun Ran, Zhongyue J Yang
Identifying function-enhancing enzyme variants is a ‘holy grail’ challenge in protein science because it will allow researchers to expand the biocatalytic toolbox for late-stage functionalization of drug-like molecules, environmental degradation of plastics and other pollutants, and medical treatment of food allergies. Data-driven strategies, including statistical modeling, machine learning, and deep
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Engineering Pseudomonas aeruginosa arylsulfatase for hydrolysis of α-configured steroid sulfates Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-09-28 Bradley J Stevenson, Andy Pranata, Malcolm D McLeod
Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is
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Affinity maturation of TCR-like antibodies using phage display guided by structural modeling Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-07-25 Rahel Frick, Lene S Høydahl, Ina Hodnebrug, Erik S Vik, Bjørn Dalhus, Ludvig M Sollid, Jeffrey J Gray, Inger Sandlie, Geir Åge Løset
TCR-like antibodies represent a unique type of engineered antibodies with specificity towards peptide:Human Leukocyte Antigen (pHLA), a ligand normally restricted to the sensitive recognition by T cells. Here, we report a phage display based sequential development path of such antibodies. The strategy goes from initial lead identification through in silico informed Complementarity Determining Region
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Cyan fluorescent proteins derived from mNeonGreen Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-04-13 Landon Zarowny, Damien Clavel, Ryan Johannson, Kévin Duarte, Hadrien Depernet, Jérôme Dupuy, Heather Baker, Alex Brown, Antoine Royant, Robert E Campbell
mNeonGreen, an engineered green fluorescent protein (GFP) derived from lancelet, is one of the most brightly fluorescent homologues of Aequorea victoria jellyfish GFP (avGFP) yet reported. In this work, we investigated whether this bright fluorescence might be retained in homologs of mNeonGreen with modified chromophore structures and altered fluorescent hues. We found mNeonGreen to be generally less
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The stability and dynamics of computationally designed proteins Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-01-15 Natali A Gonzalez, Brigitte A Li, Michelle E McCully
Protein stability, dynamics and function are intricately linked. Accordingly, protein designers leverage dynamics in their designs and gain insight to their successes and failures by analyzing their proteins’ dynamics. Molecular dynamics (MD) simulations are a powerful computational tool for quantifying both local and global protein dynamics. This review highlights studies where MD simulations were
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Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-01-06 Garima Kumar, Sarah Sterrett, Lucinda Hall, Edlue Tabengwa, Kazuhito Honjo, Michael Larimer, Randall S Davis, Paul A Goepfert, Benjamin M Larimer
Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain
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Stabilization of the SARS-CoV-2 receptor binding domain by protein core redesign and deep mutational scanning Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-01-01 Alison C Leonard,Jonathan J Weinstein,Paul J Steiner,Annette H Erbse,Sarel J Fleishman,Timothy A Whitehead
Abstract Stabilizing antigenic proteins as vaccine immunogens or diagnostic reagents is a stringent case of protein engineering and design as the exterior surface must maintain recognition by receptor(s) and antigen—specific antibodies at multiple distinct epitopes. This is a challenge, as stability enhancing mutations must be focused on the protein core, whereas successful computational stabilization
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Correction to: Protease-stable DARPins as promising oral therapeutics Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-01-01 Rudo A Simeon,Yu Zeng,Vikas Chonira,Andrea Martinez Aguirre,Mauricio Lasagna,Marko Baloh,Joseph A Sorg,Cecilia Tommos,Zhilei Chen
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Yeast surface display-based identification of ACE2 mutations that modulate SARS-CoV-2 spike binding across multiple mammalian species Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2022-01-01 Pete Heinzelman, Jonathan C Greenhalgh, Philip A Romero
Understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with different mammalian angiotensin-converting enzyme II (ACE2) cell entry receptors elucidates determinants of virus transmission and facilitates development of vaccines for humans and animals. Yeast display-based directed evolution identified conserved ACE2 mutations that increase spike binding across multiple
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Immortalization and functional screening of natively paired human T cell receptor repertoires Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-12-30 Ahmed S Fahad, Cheng-Yu Chung, Sheila N Lopez Acevedo, Nicoleen Boyle, Bharat Madan, Matias F Gutiérrez-González, Rodrigo Matus-Nicodemos, Amy D Laflin, Rukmini R Ladi, John Zhou, Jacy Wolfe, Sian Llewellyn-Lacey, Richard A Koup, Daniel C Douek, Henry H Balfour Jr, David A Price, Brandon J DeKosky
Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. We developed a new high-throughput approach
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Modifying pH-sensitive PCSK9/LDLR interactions as a strategy to enhance hepatic cell uptake of low-density lipoprotein cholesterol (LDL-C) Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-12-23 Lital Ben-Naim, Isam Khalaila, Niv Papo
LDL-receptor (LDLR)-mediated uptake of LDL-C into hepatocytes is impaired by lysosomal degradation of LDLR, which is promoted by proprotein convertase subtilisin/kexin type 9 (PCSK9). Cell surface binding of PCSK9 to LDLR produces a complex that translocates to an endosome, where the acidic pH strengthens the binding affinity of PCSK9 to LDLR, preventing LDLR recycling to the cell membrane. We present
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Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-11-22 Samantha R Summers, Sarah Alamdari, Casey J Kraft, Roman Brunecky, Jim Pfaendtner, Joel L Kaar
Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each
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Generation of a nanobody against HER2 tyrosine kinase using phage display library screening for HER2-positive breast cancer therapy development Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-11-10 Thomanai Lamtha, Lueacha Tabtimmai, Kunan Bangphoomi, Duangnapa Kiriwan, Aijaz A Malik, Wanpen Chaicumpa, Paul M P van Bergen en Henegouwen, Kiattawee Choowongkomon
Human epidermal growth factor receptor 2 (HER2) protein overexpression is found in ~30% of invasive breast carcinomas and in a high proportion of noninvasive ductal carcinomas in situ. Targeted cancer therapy is based on monoclonal antibodies and kinase inhibitors and reflects a new era of cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional
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Exploring the binding of rationally engineered tandem-repeat proteins to E3 ubiquitin ligase Keap1 Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-11-10 Sarah K Madden, Laura S Itzhaki
The process of displaying functional peptides by ‘grafting’ them onto loops of a stable protein scaffold can be used to impart binding affinity for a target, but it can be difficult to predict the affinity of the grafted peptide and the effect of grafting on scaffold stability. In this study, we show that a series of peptides that bind to the E3 ubiquitin ligase Keap1 can be grafted into the inter-repeat
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DE-STRESS: a user-friendly web application for the evaluation of protein designs Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-11-05 Michael J Stam, Christopher W Wood
De novo protein design is a rapidly growing field, and there are now many interesting and useful examples of designed proteins in the literature. However, most designs could be classed as failures when characterised in the lab, usually as a result of low expression, misfolding, aggregation or lack of function. This high attrition rate makes protein design unreliable and costly. It is possible that
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Protease-stable DARPins as promising oral therapeutics Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-10-28 Rudo A Simeon, Yu Zeng, Vikas Chonira, Andrea Martinez Aguirre, Mauricio Lasagna, Marko Baloh, Joseph A Sorg, Cecilia Tommos, Zhilei Chen
Clostridioides difficile is an enteric bacterium whose exotoxins, TcdA and TcdB, inactivate small GTPases within the host cells, leading to bloody diarrhea. In prior work, our group engineered a panel of potent TcdB-neutralizing designed ankyrin repeat proteins (DARPin) as oral therapeutics against C. difficile infection. However, all these DARPins are highly susceptible to digestion by gut-resident
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Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-10-22 Yuya Shimozawa, Tomoki Himiyama, Tsutomu Nakamura, Yoshiaki Nishiya
Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential
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Improved thermostability of proteinase K and recognizing the synergistic effect of Rosetta and FoldX approaches Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-10-09 Yang Zhao, Daixi Li, Xue Bai, Manjie Luo, Yan Feng, Yilei Zhao, Fuqiang Ma, Guang-Yu Yang
Proteinase K (PRK) is a proteolytic enzyme that has been widely used in industrial applications. However, poor stability has severely limited the uses of PRK. In this work, we used two structure-guided rational design methods, Rosetta and FoldX, to modify PRK thermostability. Fifty-two single amino acid conversion mutants were constructed based on software predictions of residues that could affect
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Rational engineering of an erythropoietin fusion protein to treat hypoxia Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-10-08 Jungmin Lee, Andyna Vernet, Nathalie G Gruber, Kasia M Kready, Devin R Burrill, Jeffrey C Way, Pamela A Silver
Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin
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A versatile assay platform for enzymatic poly(ethylene-terephthalate) degradation Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-08-24 Weigert S, Gagsteiger A, Menzel T, et al.
AbstractAccumulation of plastic and subsequent microplastic is a major environmental challenge. With the discovery of potent polyethylene terephthalate (PET)-degrading enzymes, a new perspective arose for environmental decomposition as well as technical recycling. To explore the enormous diversity of potential PET-degrading enzymes in nature and also to conveniently employ techniques like protein engineering
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Computational design and experimental optimization of protein binders with prospects for biomedical applications Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-08-26 Alessandro Bonadio, Julia M Shifman
Protein-based binders have become increasingly more attractive candidates for drug and imaging agent development. Such binders could be evolved from a number of different scaffolds, including antibodies, natural protein effectors and unrelated small protein domains of different geometries. While both computational and experimental approaches could be utilized for protein binder engineering, in this
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The design and evolution of fluorescent protein-based sensors for monoatomic ions in biology Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-08-19 Kiheon Baek, Ke Ji, Weicheng Peng, Sureshee M Liyanaarachchi, Sheel C Dodani
Living cells rely on a finely tuned symphony of inorganic ion gradients composed of both cations and anions. This delicate balance is maintained by biological receptors all acting in concert to selectively recognize and position ions for homeostasis. These dynamic processes can be intercepted and visualized with optical microscopy at the organismal, tissue, cellular and subcellular levels using fluorescent
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Machine learning for enzyme engineering, selection and design Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-07-23 Ryan Feehan, Daniel Montezano, Joanna S G Slusky
Machine learning is a useful computational tool for large and complex tasks such as those in the field of enzyme engineering, selection and design. In this review, we examine enzyme-related applications of machine learning. We start by comparing tools that can identify the function of an enzyme and the site responsible for that function. Then we detail methods for optimizing important experimental
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Engineering stable carbonic anhydrases for CO2 capture: a critical review Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-07-20 Mirfath Sultana Mesbahuddin, Aravindhan Ganesan, Subha Kalyaanamoorthy
In the search for green CO2-capture technology to combat global warming, bioengineering of carbonic anhydrases (CAs) is being sought for with target adaptabilities of extreme temperatures and alkaline pH conditions. The modern in silico screening of protein engineering complements the conventional in vitro high-throughput via generation of iteratively cumulating e-library of diverse beneficial mutations
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Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-07-14 Antonija Marjanovic, Carlos J Ramírez-Palacios, Marcelo F Masman, Jeroen Drenth, Marleen Otzen, Siewert-Jan Marrink, Dick B Janssen
Diaminopimelate decarboxylases (DAPDCs) are highly selective enzymes that catalyze the common final step in different lysine biosynthetic pathways, i.e. the conversion of meso-diaminopimelate (DAP) to L-lysine. We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since
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Generation of a 100-billion cyclic peptide phage display library having a high skeletal diversity Protein Eng. Des. Sel. (IF 2.4) Pub Date : 2021-06-30 Vanessa Carle, Xu-Dong Kong, Alice Comberlato, Chelsea Edwards, Cristina Díaz-Perlas, Christian Heinis
Phage display is a powerful technique routinely used for the generation of peptide- or protein-based ligands. The success of phage display selections critically depends on the size and structural diversity of the libraries, but the generation of large libraries remains challenging. In this work, we have succeeded in developing a phage display library comprising around 100 billion different (bi)cyclic