The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Uwe T Bornscheuer - One of the best experts on this subject based on the ideXlab platform.
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conversion of a mono and diacylglycerol lipase into a triacylglycerol lipase by Protein Engineering
ChemBioChem, 2015Co-Authors: Grzegorz M Popowicz, Ioannis V Pavlidis, Uwe T Bornscheuer, Pengfei Zhou, Yonghua WangAbstract:: Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono- and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By Protein Engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild-type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity.
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discovery application and Protein Engineering of baeyer villiger monooxygenases for organic synthesis
Organic and Biomolecular Chemistry, 2012Co-Authors: Kathleen Balke, Maria Kadow, Hendrik Mallin, Stefan Sas, Uwe T BornscheuerAbstract:Baeyer–Villiger monooxygenases (BVMOs) are useful enzymes for organic synthesis as they enable the direct and highly regio- and stereoselective oxidation of ketones to esters or lactones simply with molecular oxygen. This contribution covers novel concepts such as searching in Protein sequence databases using distinct motifs to discover new Baeyer–Villiger monooxygenases as well as high-throughput assays to facilitate Protein Engineering in order to improve BVMOs with respect to substrate range, enantioselectivity, thermostability and other properties. Recent examples for the application of BVMOs in synthetic organic synthesis illustrate the broad potential of these biocatalysts. Furthermore, methods to facilitate the more efficient use of BVMOs in organic synthesis by applying e.g. improved cofactor regeneration, substrate feed and in situ product removal or immobilization are covered in this perspective.
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Protein Engineering of α β hydrolase fold enzymes
ChemInform, 2011Co-Authors: Helge Jochens, Romas J Kazlauskas, Martin Hesseler, Konstanze Stiba, Santosh Kumar Padhi, Uwe T BornscheuerAbstract:The superfamily of α/β-hydrolase fold enzymes is one of the largest known Protein families, including a broad range of synthetically useful enzymes such as lipases, esterases, amidases, hydroxynitrile lyases, epoxide hydrolases and dehalogenases. This minireview covers methods developed for efficient Protein Engineering of these enzymes. Special emphasis is placed on the alteration of enzyme properties such as substrate range, thermostability and enantioselectivity for their application in biocatalysis. In addition, concepts for the investigation of the evolutionary relationship between the different members of this Protein superfamily are covered, together with successful examples.
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the alpha beta hydrolase fold 3dm database abhdb as a tool for Protein Engineering
ChemBioChem, 2010Co-Authors: Robert Kourist, Helge Jochens, Santosh Kumar Padhi, Sebastian Bartsch, Remko Kuipers, Markus Gall, Dominique Bottcher, Henkjan Joosten, Uwe T BornscheuerAbstract:Aligning the haystack to expose the needle: The 3DM method was used to generate a comprehensive database of the a/s-hydrolase fold enzyme superfamily. This database facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made. In addition high-quality libraries for Protein Engineering can be easily designed.
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Finding better Protein Engineering strategies
Nature Chemical Biology, 2009Co-Authors: Romas J Kazlauskas, Uwe T BornscheuerAbstract:Protein improvement strategies today involve widely varying combinations of rational design with random mutagenesis and screening. To make further progress—defined as making subsequent Protein Engineering problems easier to solve—Protein engineers must critically compare these strategies and eliminate less effective ones.
Julio M Fernandez - One of the best experts on this subject based on the ideXlab platform.
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mechanical design of Proteins studied by single molecule force spectroscopy and Protein Engineering
Progress in Biophysics & Molecular Biology, 2000Co-Authors: Mariano Carrionvazquez, Andres F Oberhauser, Thomas E Fisher, Piotr E Marszalek, Hongbin Li, Julio M FernandezAbstract:Mechanical unfolding and refolding may regulate the molecular elasticity of modular Proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein Engineering techniques allow the construction of homomeric polyProteins for the precise analysis of the mechanical unfolding of single domains. a-Helical domains are mechanically compliant, whereas b-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in Proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of Proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp). # 2000 Elsevier Science Ltd. All rights reserved.
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mechanical design of Proteins studied by single molecule force spectroscopy and Protein Engineering
Progress in Biophysics & Molecular Biology, 2000Co-Authors: Mariano Carrionvazquez, Andres F Oberhauser, Thomas E Fisher, Piotr E Marszalek, Julio M FernandezAbstract:Mechanical unfolding and refolding may regulate the molecular elasticity of modular Proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein Engineering techniques allow the construction of homomeric polyProteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in Proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of Proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).
George M Church - One of the best experts on this subject based on the ideXlab platform.
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low n Protein Engineering with data efficient deep learning
Nature Methods, 2021Co-Authors: Surojit Biswas, Ethan C Alley, Grigory Khimulya, George M Church, Kevin M EsveltAbstract:Protein Engineering has enormous academic and industrial potential. However, it is limited by the lack of experimental assays that are consistent with the design goal and sufficiently high throughput to find rare, enhanced variants. Here we introduce a machine learning-guided paradigm that can use as few as 24 functionally assayed mutant sequences to build an accurate virtual fitness landscape and screen ten million sequences via in silico directed evolution. As demonstrated in two dissimilar Proteins, GFP from Aequorea victoria (avGFP) and E. coli strain TEM-1 β-lactamase, top candidates from a single round are diverse and as active as engineered mutants obtained from previous high-throughput efforts. By distilling information from natural Protein sequence landscapes, our model learns a latent representation of 'unnaturalness', which helps to guide search away from nonfunctional sequence neighborhoods. Subsequent low-N supervision then identifies improvements to the activity of interest. In sum, our approach enables efficient use of resource-intensive high-fidelity assays without sacrificing throughput, and helps to accelerate engineered Proteins into the fermenter, field and clinic.
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low n Protein Engineering with data efficient deep learning
bioRxiv, 2020Co-Authors: Surojit Biswas, Ethan C Alley, Grigory Khimulya, George M Church, Kevin M EsveltAbstract:Abstract Protein Engineering has enormous academic and industrial potential. However, it is limited by the lack of experimental assays that are consistent with the design goal and sufficiently high-throughput to find rare, enhanced variants. Here we introduce a machine learning-guided paradigm that can use as few as 24 functionally assayed mutant sequences to build an accurate virtual fitness landscape and screen ten million sequences via in silico directed evolution. As demonstrated in two highly dissimilar Proteins, avGFP and TEM-1 β-lactamase, top candidates from a single round are diverse and as active as engineered mutants obtained from previous multi-year, high-throughput efforts. Because it distills information from both global and local sequence landscapes, our model approximates Protein function even before receiving experimental data, and generalizes from only single mutations to propose high-functioning epistatically non-trivial designs. With reproducible >500% improvements in activity from a single assay in a 96-well plate, we demonstrate the strongest generalization observed in machine-learning guided Protein design to date. Taken together, our approach enables efficient use of resource intensive high-fidelity assays without sacrificing throughput. By encouraging alignment with endpoint objectives, low-N design will accelerate engineered Proteins into the fermenter, field, and clinic.
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unified rational Protein Engineering with sequence based deep representation learning
Nature Methods, 2019Co-Authors: Ethan C Alley, Grigory Khimulya, Surojit Biswas, Mohammed Alquraishi, George M ChurchAbstract:Rational Protein Engineering requires a holistic understanding of Protein function. Here, we apply deep learning to unlabeled amino-acid sequences to distill the fundamental features of a Protein into a statistical representation that is semantically rich and structurally, evolutionarily and biophysically grounded. We show that the simplest models built on top of this unified representation (UniRep) are broadly applicable and generalize to unseen regions of sequence space. Our data-driven approach predicts the stability of natural and de novo designed Proteins, and the quantitative function of molecularly diverse mutants, competitively with the state-of-the-art methods. UniRep further enables two orders of magnitude efficiency improvement in a Protein Engineering task. UniRep is a versatile summary of fundamental Protein features that can be applied across Protein Engineering informatics.
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unified rational Protein Engineering with sequence only deep representation learning
bioRxiv, 2019Co-Authors: Ethan C Alley, Grigory Khimulya, Surojit Biswas, Mohammed Alquraishi, George M ChurchAbstract:Abstract Rational Protein Engineering requires a holistic understanding of Protein function. Here, we apply deep learning to unlabelled amino acid sequences to distill the fundamental features of a Protein into a statistical representation that is semantically rich and structurally, evolutionarily, and biophysically grounded. We show that the simplest models built on top of this unified representation (UniRep) are broadly applicable and generalize to unseen regions of sequence space. Our data-driven approach reaches near state-of-the-art or superior performance predicting stability of natural and de novo designed Proteins as well as quantitative function of molecularly diverse mutants. UniRep further enables two orders of magnitude cost savings in a Protein Engineering task. We conclude UniRep is a versatile Protein summary that can be applied across Protein Engineering informatics.
Tom W Muir - One of the best experts on this subject based on the ideXlab platform.
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Protein Engineering through tandem transamidation
Nature Chemistry, 2019Co-Authors: Robert E Thompson, Adam J. Stevens, Tom W MuirAbstract:Semisynthetic Proteins engineered to contain non-coded elements such as post-translational modifications (PTMs) represent a powerful class of tools for interrogating biological processes. Here, we introduce a one-pot, chemoenzymatic method that allows broad access to chemically modified Proteins. The approach involves a tandem transamidation reaction cascade that integrates intein-mediated Protein splicing with enzyme-mediated peptide ligation. We show that this approach can be used to introduce PTMs and biochemical probes into a range of Proteins including Cas9 nuclease and the transcriptional regulator MeCP2, which causes Rett syndrome when mutated. The versatility of the approach is further illustrated through the chemical tailoring of histone Proteins within a native chromatin setting. We expect our approach will extend the scope of semisynthesis in Protein Engineering. A method for Engineering chemically modified Proteins has now been developed using a chemoenzymatic cascade of sortase-mediated transpeptidation and Protein trans-splicing. Using this one-pot approach enabled the generation of site-specifically modified Proteins in vitro and in isolated cell nuclei.
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A promiscuous split intein with expanded Protein Engineering applications
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Adam J. Stevens, David Cowburn, Neel H. Shah, Giridhar Sekar, Anahita Z. Mostafavi, Tom W MuirAbstract:The Protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an “extein dependence,” whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided Protein Engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting “promiscuous” inteins are shown to be superior reagents for Protein cyclization and Protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins.
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expressed Protein ligation a general method for Protein Engineering
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Tom W Muir, Dolan Sondhi, Philip A ColeAbstract:A Protein semisynthesis method—expressed Protein ligation—is described that involves the chemoselective addition of a peptide to a recombinant Protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the Protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant Protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the Protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed Protein ligation is a simple and powerful new method in Protein Engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into Proteins of any size.
Mariano Carrionvazquez - One of the best experts on this subject based on the ideXlab platform.
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mechanical design of Proteins studied by single molecule force spectroscopy and Protein Engineering
Progress in Biophysics & Molecular Biology, 2000Co-Authors: Mariano Carrionvazquez, Andres F Oberhauser, Thomas E Fisher, Piotr E Marszalek, Hongbin Li, Julio M FernandezAbstract:Mechanical unfolding and refolding may regulate the molecular elasticity of modular Proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein Engineering techniques allow the construction of homomeric polyProteins for the precise analysis of the mechanical unfolding of single domains. a-Helical domains are mechanically compliant, whereas b-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in Proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of Proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp). # 2000 Elsevier Science Ltd. All rights reserved.
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mechanical design of Proteins studied by single molecule force spectroscopy and Protein Engineering
Progress in Biophysics & Molecular Biology, 2000Co-Authors: Mariano Carrionvazquez, Andres F Oberhauser, Thomas E Fisher, Piotr E Marszalek, Julio M FernandezAbstract:Mechanical unfolding and refolding may regulate the molecular elasticity of modular Proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein Engineering techniques allow the construction of homomeric polyProteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in Proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of Proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).
