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1.
Exploring Protein Supersecondary Structure Through Changes in Protein Folding, Stability, and Flexibility.
Pires, DEV, Rodrigues, CHM, Albanaz, ATS, Karmakar, M, Myung, Y, Xavier, J, Michanetzi, EM, Portelli, S, Ascher, DB
Methods in molecular biology (Clifton, N.J.). 2019;:173-185
Abstract
The ability to predict how mutations affect protein structure, folding, and flexibility can elucidate the molecular mechanisms leading to disruption of supersecondary structures, the emergence of phenotypes, as well guiding rational protein engineering. The advent of fast and accurate computational tools has enabled us to comprehensively explore the landscape of mutation effects on protein structures, prioritizing mutations for rational experimental validation.Here we describe the use of two complementary web-based in silico methods, DUET and DynaMut, developed to infer the effects of mutations on folding, stability, and flexibility and how they can be used to explore and interpret these effects on protein supersecondary structures.
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2.
QTY code designed thermostable and water-soluble chimeric chemokine receptors with tunable ligand affinity.
Qing, R, Han, Q, Skuhersky, M, Chung, H, Badr, M, Schubert, T, Zhang, S
Proceedings of the National Academy of Sciences of the United States of America. 2019;(51):25668-25676
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Abstract
Chemokine receptors are of great interest as they play a critical role in many immunological and pathological processes. The ability to study chemokine receptors in aqueous solution without detergent would be significant because natural receptors require detergents to become soluble. We previously reported using the QTY code to design detergent-free chemokine receptors. We here report the design of 2 detergent-free chimeric chemokine receptors that were experimentally unattainable in detergent solution. We designed chimeric receptors by switching the N terminus and 3 extracellular (EC) loops between different receptors. Specifically, we replaced the N terminus and 3 EC loops of CCR5QTY with the N terminus and 3 EC loops of CXCR4. The ligand for CXCR4; namely CXCL12, binds to the chimeric receptor CCR5QTY (7TM)-CXCR4 (N terminus+3 EC loops), but with lower affinity compared to CXCR4; the CCL5 ligand of CCR5 binds the chimeric receptor with ∼20× lower affinity. The chimeric design helps to elucidate the mechanism of native receptor-ligand interaction. We also show that all detergent-free QTY-designed chemokine receptors, expressed in Escherichia coli, bind to their respective chemokines with affinities in the nanomolar (nM) range, similar to the affinities of native receptors and SF9-produced QTY variants. These QTY-designed receptors exhibit remarkable thermostability in the presence of arginine and retain ligand-binding activity after heat treatment at 60 °C for 4 h and 24 h, and at 100 °C for 10 min. Our design approach enables affordable scale-up production of detergent-free QTY variant chemokine receptors with tunable functionality for various uses.
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Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.
Hatlem, D, Trunk, T, Linke, D, Leo, JC
International journal of molecular sciences. 2019;(9)
Abstract
The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.
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Current Trends in Protein Engineering: Updates and Progress.
Sinha, R, Shukla, P
Current protein & peptide science. 2019;(5):398-407
Abstract
Proteins are one of the most important and resourceful biomolecules that find applications in health, industry, medicine, research, and biotechnology. Given its tremendous relevance, protein engineering has emerged as significant biotechnological intervention in this area. Strategic utilization of protein engineering methods and approaches has enabled better enzymatic properties, better stability, increased catalytic activity and most importantly, interesting and wide range applicability of proteins. In fact, the commercialization of engineered proteins have manifested in economically beneficial and viable solutions for industry and healthcare sector. Protein engineering has also evolved to become a powerful tool contributing significantly to the developments in both synthetic biology and metabolic engineering. The present review revisits the current trends in protein engineering approaches such as rational design, directed evolution, de novo design, computational approaches etc. and encompasses the recent progresses made in this field over the last few years. The review also throws light on advanced or futuristic protein engineering aspects, which are being explored for design and development of novel proteins with improved properties or advanced applications.
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5.
Near-Infrared Fluorescent Proteins and Their Applications.
Karasev, MM, Stepanenko, OV, Rumyantsev, KA, Turoverov, KK, Verkhusha, VV
Biochemistry. Biokhimiia. 2019;(Suppl 1):S32-S50
Abstract
High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650-900 nm) open a possibility for in vivo imaging of biological processes at the micro- and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes - natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.
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Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation.
Zeuner, B, Teze, D, Muschiol, J, Meyer, AS
Molecules (Basel, Switzerland). 2019;(11)
Abstract
Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite -1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite -1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.
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Machine Learning Applied to Predicting Microorganism Growth Temperatures and Enzyme Catalytic Optima.
Li, G, Rabe, KS, Nielsen, J, Engqvist, MKM
ACS synthetic biology. 2019;(6):1411-1420
Abstract
Enzymes that catalyze chemical reactions at high temperatures are used for industrial biocatalysis, applications in molecular biology, and as highly evolvable starting points for protein engineering. The optimal growth temperature (OGT) of organisms is commonly used to estimate the stability of enzymes encoded in their genomes, but the number of experimentally determined OGT values are limited, particularly for thermophilic organisms. Here, we report on the development of a machine learning model that can accurately predict OGT for bacteria, archaea, and microbial eukaryotes directly from their proteome-wide 2-mer amino acid composition. The trained model is made freely available for reuse. In a subsequent step we use OGT data in combination with amino acid composition of individual enzymes to develop a second machine learning model-for prediction of enzyme catalytic temperature optima ( Topt). The resulting model generates enzyme Topt estimates that are far superior to using OGT alone. Finally, we predict Topt for 6.5 million enzymes, covering 4447 enzyme classes, and make the resulting data set available to researchers. This work enables simple and rapid identification of enzymes that are potentially functional at extreme temperatures.
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Recent Progress in Chemical Modification of Proteins.
Sakamoto, S, Hamachi, I
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry. 2019;(1):5-27
Abstract
Chemical modification of proteins is important for creating a myriad of engineered proteins and for elucidating the function and dynamics of proteins in live cells. A wide variety of chemical protein modification methods have been developed and can be categorized into three classes: (i) modification of proteins using the reactivity of naturally occurring amino acids; (ii) modification by bioorthogonal reactions using unnatural amino acids, most of which can be site-selectively incorporated into proteins-of-interest using genetic codon expansion techniques; and (iii) recognition driven chemical modification, which is the only approach that allows modification of endogenous proteins without any genetic manipulation even under heavily crowded and multi-molecular conditions, as in live cells and organisms. All of these approaches have merits and limitations. In this review, we summarize these approaches and discuss their characteristics with respect to specificity, reaction rate and versatility.
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9.
Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases.
Kim, D, Luk, K, Wolfe, SA, Kim, JS
Annual review of biochemistry. 2019;:191-220
Abstract
Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.
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10.
Constructive approach for synthesis of a functional IgG using a reconstituted cell-free protein synthesis system.
Murakami, S, Matsumoto, R, Kanamori, T
Scientific reports. 2019;(1):671
Abstract
IgG is an indispensable biological experimental tool as well as a widely-used therapeutic protein. However, cell culture-based expression of monoclonal IgG is costly and time-consuming, making this process difficult to use for high-throughput screening in early-stage evaluation of biologics. With the goal of establishing a fast, simple, and robust high-throughput expression system for IgG, we implemented the synthesis of functional aglycosylated IgG by constructive approach based on a reconstituted prokaryotic cell-free protein synthesis system (PURE system). Optimization of the PURE system revealed that the following factors and reaction conditions were needed for IgG synthesis: (1) inclusion of the disulfide bond isomerase DsbC, (2) adjustment of the GSH/GSSG ratio, (3) inclusion of the molecular chaperone DnaK and its cofactors, and (4) use of an extended incubation time. Synthesis temperature and template DNA ratio (light chain-/heavy chain-encoding) also had been optimized for each IgG. Under optimal conditions, peak production of the anti-HER2 antibody trastuzumab reached 124 µg/mL. Furthermore, the active forms of other IgGs, including IgG1, IgG2, and IgG4 subclasses, also were synthesized. These results provide basic information for the development of novel high-throughput expression and functional screening systems for IgG, as well as useful information for understanding the IgG synthesis process.