Engineering Brassica Crops to Optimize Delivery of Bioactive Products Postcooking
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59 BASIC BIOLOGICAL SCIENCES↗
SEARCH · Engineering Papers
Results for “thermostable proteins”
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Self-driving laboratories to autonomously navigate the protein fitness landscape
Abstract Protein engineering has nearly limitless applications across chemistry, energy and medicine, but creating new proteins with improved or novel functions remains slow, labor-intensive and inefficient. Here we present the Self-driving Autonomous Machines for Protein Landscape Exploration (SAMPLE) platform for fully autonomous protein engineering. SAMPLE is driven by an intelligent agent that learns protein sequence–function relationships, designs new proteins and sends designs to a fully automated robotic system that experimentally tests the designed proteins and provides feedback to improve the agent’s understanding of the system. We deploy four SAMPLE agents with the goal of engineering glycoside hydrolase enzymes with enhanced thermal tolerance. Despite showing individual differences in their search behavior, all four agents quickly converge on thermostable enzymes. Self-driving laboratories automate and accelerate the scientific discovery process and hold great potential for the fields of protein engineering and synthetic biology.
Rapp, Jacob T. (ORCID:0000000226835204)↗
Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization
Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether “staples” into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with pchloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔT m ' = +27 °C) and temperature-induced heme loss (ΔT 50 = +30 °C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallographic analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Computational design of highly signalling-active membrane receptors through solvent-mediated allosteric networks
Abstract Protein catalysis and allostery require the atomic-level orchestration and motion of residues and ligand, solvent and protein effector molecules. However, the ability to design protein activity through precise protein–solvent cooperative interactions has not yet been demonstrated. Here we report the design of 14 membrane receptors that catalyse G protein nucleotide exchange through diverse engineered allosteric pathways mediated by cooperative networks of intraprotein, protein–ligand and –solvent molecule interactions. Consistent with predictions, the designed protein activities correlated well with the level of plasticity of the networks at flexible transmembrane helical interfaces. Several designs displayed considerably enhanced thermostability and activity compared with related natural receptors. The most stable and active variant crystallized in an unforeseen signalling-active conformation, in excellent agreement with the design models. The allosteric network topologies of the best designs bear limited similarity to those of natural receptors and reveal an allosteric interaction space larger than previously inferred from natural proteins. The approach should prove useful for engineering proteins with novel complex protein binding, catalytic and signalling activities.
Chemistry↗
Simultaneous enhancement of multiple functional properties using evolution-informed protein design
Abstract A major challenge in protein design is to augment existing functional proteins with multiple property enhancements. Altering several properties likely necessitates numerous primary sequence changes, and novel methods are needed to accurately predict combinations of mutations that maintain or enhance function. Models of sequence co-variation (e.g., EVcouplings), which leverage extensive information about various protein properties and activities from homologous protein sequences, have proven effective for many applications including structure determination and mutation effect prediction. We apply EVcouplings to computationally design variants of the model protein TEM-1 β -lactamase. Nearly all the 14 experimentally characterized designs were functional, including one with 84 mutations from the nearest natural homolog. The designs also had large increases in thermostability, increased activity on multiple substrates, and nearly identical structure to the wild type enzyme. This study highlights the efficacy of evolutionary models in guiding large sequence alterations to generate functional diversity for protein design applications.
59 BASIC BIOLOGICAL SCIENCES↗
Ordered nanoparticle arrays formed on engineered chaperonin protein templates
Traditional methods for fabricating nanoscale arrays are usually based on lithographic techniques. Alternative new approaches rely on the use of nanoscale templates made of synthetic or biological materials. Some proteins, for example, have been used to form ordered two-dimensional arrays. Here, we fabricated nanoscale ordered arrays of metal and semiconductor quantum dots by binding preformed nanoparticles onto crystalline protein templates made from genetically engineered hollow double-ring structures called chaperonins. Using structural information as a guide, a thermostable recombinant chaperonin subunit was modified to assemble into chaperonins with either 3 nm or 9 nm apical pores surrounded by chemically reactive thiols. These engineered chaperonins were crystallized into two-dimensional templates up to 20 microm in diameter. The periodic solvent-exposed thiols within these crystalline templates were used to size-selectively bind and organize either gold (1.4, 5 or 10nm) or CdSe-ZnS semiconductor (4.5 nm) quantum dots into arrays. The order within the arrays was defined by the lattice of the underlying protein crystal. By combining the self-assembling properties of chaperonins with mutations guided by structural modelling, we demonstrate that quantum dots can be manipulated using modified chaperonins and organized into arrays for use in next-generation electronic and photonic devices.
Chaperonins/chemistry/ultrastructure↗
Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline
Abstract As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.
36 MATERIALS SCIENCE↗
Modular adjuvant-free pan-HLA-DR-immunotargeting subunit vaccine against SARS-CoV-2 elicits broad sarbecovirus-neutralizing antibody responses
Subunit vaccines typically require co-administration with an adjuvant to elicit protective immunity, adding development hurdles that can impede rapid pandemic responses. To circumvent the need for adjuvant in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine, we engineer a thermostable immunotargeting vaccine (ITV) that leverages the pan-HLA-DR monoclonal antibody 44H10 to deliver the viral spike protein receptor-binding domain (RBD) to antigen-presenting cells. X-ray crystallography shows that 44H10 binds to a conserved epitope on HLA-DR, providing the basis for its broad HLA-DR reactivity. Adjuvant-free ITV immunization in rabbits and ferrets induces robust anti-RBD antibody responses that neutralize SARS-CoV-2 variants of concern and protect recipients from SARS-CoV-2 challenge. We demonstrate that the modular nature of the ITV scaffold with respect to helper T cell epitopes and diverse RBD antigens facilitates broad sarbecovirus neutralization. Our findings support anti-HLA-DR immunotargeting as an effective means to induce strong antibody responses to subunit antigens without requiring an adjuvant.
60 APPLIED LIFE SCIENCES↗
Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens
The gene encoding an extracellular alpha-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.
Bernhardsdotter, Eva C. M. J.↗
Cloning and Characterization of an Alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens
The gene encoding an extracellular a-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.
Bernhardsdotter, Eva C. M. J.↗
Dairy manure acidogenic fermentation at hyperthermophilic temperature enabled superior activity of thermostable hydrolytic enzymes linked to the genus Caldicoprobacter
In this study, fermentation experiments were conducted under mesophilic, thermophilic, and hyperthermophilic conditions to investigate adaptation of microbial communities and its effect on extracellular enzyme activities toward degradation of cellulose, hemicellulose and proteins in dairy manure. Here, hyperthermophilic conditions transformed the microbiome structure and stimulated activity of extracellular proteolytic, cellulolytic, and hemicellulolytic enzymes. Specifically, the activities of protease, cellulose 1,4-β-cellobiosidase, and β-glucosidase secreted by hyperthermophilic microbes were higher by 22%, 47% and 49% compared to those produced by mesophilic and thermophilic communities. Enhanced hydrolytic activity of hyperthermophilic microbes enabled improved feedstock solubilization and production of 39% and 22% more soluble COD than mesophilic and thermophilic microbes, respectively. Connections between hydrolytic function and microbial community structure at various temperatures were assessed using the PICRUSt2 computational tool. Genus Caldicoprobacter was identified as the primary candidate responsible for increased production of thermostable endo-1,4-β-glucanase, β-glucosidase and endo-1,4-β-xylanase, and enhanced hydrolytic performance of hyperthermophilic microbial community.
09 BIOMASS FUELS↗
Increasing thermostability of the key photorespiratory enzyme glycerate 3‐kinase by structure‐based recombination
As global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.
59 BASIC BIOLOGICAL SCIENCES↗
Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
59 BASIC BIOLOGICAL SCIENCES↗
Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines
Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1 C ) regions to GP metastability. Specific stalk and HR1 C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1 C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.
60 APPLIED LIFE SCIENCES↗
An Arabinoxylan Extracted from Corn Grain Is Inhibitory to Cellulase Activity
The maize seed expression system is a powerful platform for recombinant protein production. It has been utilized for high-yield production of industrial cellulases such as endo-1,4-b-Dglucanase (E1) and 1,4-b-D-glucan cellobiohydrolases (CBHI & CBHII). However, inhibitory compounds that could significantly reduce the cellulolytic efficiency were detected even in purified E1 and CBHII samples. After several steps of purification and analysis, the inhibitory compound found in the purified E1 sample had characteristics consistent with a thermostable polysaccharide with a molecular size over 10 kDa. Further polysaccharide analysis confirmed the molecular size of the detected polysaccharide and suggested that it is an arabinoxylan with a high arabinose/xylose ratio. Even though the strict column purification process should have removed water soluble non-protein molecules during protein purification, the inhibitor remained. Here, a possible explanation is that recombinant cellulase can bind to the inhibitor, carry it through all purification steps and finally release it when denatured by heat.
59 BASIC BIOLOGICAL SCIENCES↗
Activation dynamics of a water-soluble human mu-opioid receptor
The mu-opioid receptor (MOR), a class A G protein-coupled receptor mediates opioid analgesia and remains a central target for pain therapeutics. While crystal structures of MOR exist, they provide limited insight into the receptor’s dynamic conformational landscape underlying function. Here, we engineered a thermostable water-soluble MOR variant (wsMOR) that retains native-like ligand-binding and activation dynamics. This variant enables high-yield production and detailed solution-phase structural studies that are challenging with membrane-embedded MOR, providing a valuable tool for studying receptor activation and aqueous-phase drug screening. Using a combined computational and experimental approach, we performed long-timescale all-atom molecular dynamics simulations together with neutron scattering and single-molecule FRET, revealing a structurally stable receptor with a diverse ensemble of conformations at different temporal resolutions. In the ligand-free state, wsMOR displayed high conformational flexibility, which decreased upon agonist binding, particularly in transmembrane helix 6, a hallmark of G protein-coupled receptor activation. Positive allosteric modulation and G protein binding further stabilized active-like states. These findings highlight wsMOR’s conformational plasticity across picosecond to millisecond timescales and provide a foundation for structure-guided development of next-generation opioid ligands with improved efficacy and safety.
E, Agyemang [University of Tennessee Knoxville]↗
Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP + -binding site. Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP + and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP + binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PD def ). We first solved the crystal structure for G6PD K403Q , a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP + binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PD def ), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PD K403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PD def and provide a foundation for future drug discovery efforts.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Enzyme property prediction using artificial intelligence
Artificial intelligence (AI)-driven enzyme property prediction enables rapid discovery and engineering of enzymes for a wide range of biotechnological and therapeutic applications. Here, we first introduce the key components in AI model development, including enzyme datasets, protein representation methods, and model architectures. We then highlight a variety of AI tools developed for the prediction of enzyme properties and functional annotations, including enzyme structure, kinetic parameters, substrate specificity, thermostability, solubility, Enzyme Commission number, and Gene Ontology term. Moreover, we describe representative downstream applications enabled by these AI tools. Finally, we discuss some challenges and opportunities as well as future prospects.
Yuan, Le [University of Illinois at Urbana-Champai↗