Combining Protein Engineering Research Articles

All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2′-fucosyllactose (2′-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2′-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2′-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2′-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2′-FL production. Therefore, to decrease the formation of byproduct 2′-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2′-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154–171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154–171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2′-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2′-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2′-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

Integrating Chemocatalysis and Enzyme Engineering for Coproduction of Bioplastic Precursors Butanediol and Succinic Acid from Xylose

AbstractPolybutylene succinate (PBS) is one of the most important biobased plastics, based on the amount produced. Owing to its high melting point and resemblance to petroleum‐based plastics (i. e. PP), PBS becomes one of the emerging bioplastics with an array of applications. PBS is manufactured by polymerization of 1,4‐butanediol and succinic acid. Thus, it is of great importance to ensure the use of renewable resources to produce the PBS precursors. As the second most abundant carbohydrate monomer on Earth, D‐xylose will be a suitable candidate for this purpose. In this work, we combined protein engineering with chemical oxidation by gold catalyst to enable transformation of D‐xylose to 1,4‐butanediol and succinic acid simultaneously. In silico docking studies and semi rational design were employed to create variants of the key enzyme, branched chain α‐keto acid decarboxylase (KdcA) with higher affinity for the intermediates in the production of 1,4‐butanediol and succinic acid. Direct enzymatic biotransformation would result in a production of both monomers with 3:1 ratio, thus not readily suitable for a direct polymerization to PBS. By developing a one‐pot multi‐step chemo‐enzymatic approach with a gold catalyst to perform the first oxidation step, we could achieve a final product ratio of 1:1. Application of an engineered KdcA variant allowed us to achieve >98% yield after four hours transformation. In contrast, after 24 h transformation, >10% intermediate was still observed when the original variant was used. We anticipate this new approach could serve as an alternative route for biotechnological productions of PBS and its precursors.

Using quantitative single molecule localization microscopy to optimize multivalent HER2-targeting ligands.

The progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling. By combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells. We detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases. Collectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics.

Phenylalanine ammonia-lyases: combining protein engineering and natural diversity.

In this study, rational design and saturation mutagenesis efforts for engineering phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) provided tailored PALs active towards challenging, highly valuable di-substituted substrates, such as the L-DOPA precursor 3,4-dimethoxy-L-phenylalanine or the 3-bromo-4-methoxy-phenylalanine. The rational design approach and saturation mutagenesis strategy unveiled identical PcPAL variants of improved activity, highlighting the limited mutational variety of the substrate specificity-modulator residues, L134, F137, I460 of PcPAL. Due to the restricted catalytic efficiency of the best performing L134A/I460V and F137V/I460V PcPAL variants, we imprinted these beneficial mutations to PALs of different origins. The variants of PALs from Arabidopsis thaliana (AtPAL) and Anabaena variabilis (AvPAL) showed higher catalytic efficiency than their PcPAL homologues. Further, the engineered PALs were also compared in terms of catalytic efficiency with a novel aromatic ammonia-lyase from Loktanella atrilutea (LaAAL), close relative of the metagenome-derived aromatic ammonia-lyase AL-11, reported recently to possess atypically high activity towards substrates with electron-donor aromatic substituents. Indeed, LaAAL outperformed the engineered Pc/At/AvPALs in the production of 3,4-dimethoxy-L-phenylalanine; however, in case of3-bromo-4-methoxy derivatives it showed no activity, with computational results supporting the occurrence of steric hindrance. Transferring the unique array of selectivity modulator residues from LaAAL to the well-characterized PALs did not enhance their activity towards the targeted substrates. Moreover, applying the rational design strategy valid for these well-characterized PALs to LaAAL decreased its activity. These results suggest that distinct tailoring rationale is required for LaAAL/AL-11-like aromatic ammonia-lyases, which might represent a distinct PAL subclass, with natural reaction and substrate scope modified through evolutionary processes. KEY POINTS: • PAL-activity for challenging substrates generated by protein engineering • Rational/semi-rational protein engineering reveals constrained mutational variability • Engineered PALs are outperformed by novel ALs of distinct catalytic site signature.

New insights into controlling radical migration pathways in heme enzymes gained from the study of a dye-decolorising peroxidase.

In heme enzymes, such as members of the dye-decolorising peroxidase (DyP) family, the formation of the highly oxidising catalytic Fe(iv)-oxo intermediates following reaction with hydrogen peroxide can lead to free radical migration (hole hopping) from the heme to form cationic tyrosine and/or tryptophan radicals. These species are highly oxidising (∼1 V vs. NHE) and under certain circumstances can catalyse the oxidation of organic substrates. Factors that govern which specific tyrosine or tryptophan the free radical migrates to in heme enzymes are not well understood, although in the case of tyrosyl radical formation the nearby proximity of a proton acceptor is a recognised facilitating factor. By using an A-type member of the DyP family (DtpAa) as an exemplar, we combine protein engineering, X-ray crystallography, hole-hopping calculations, EPR spectroscopy and kinetic modelling to provide compelling new insights into the control of radical migration pathways following reaction of the heme with hydrogen peroxide. We demonstrate that the presence of a tryptophan/tyrosine dyad motif displaying a T-shaped orientation of aromatic rings on the proximal side of the heme dominates the radical migration landscape in wild-type DtpAa and continues to do so following the rational engineering into DtpAa of a previously identified radical migration pathway in an A-type homolog on the distal side of the heme. Only on disrupting the proximal dyad, through removal of an oxygen atom, does the radical migration pathway then switch to the engineered distal pathway to form the desired tyrosyl radical. Implications for protein design and biocatalysis are discussed.

Enhancing Trans-Nerolidol Productivity in Yarrowia lipolytica by Improving Precursor Supply and Optimizing Nerolidol Synthase Activity.

The low enzymatic capability of terpene synthases and the limited availability of precursors often hinder the productivity of terpenes in microbial hosts. Herein, a systematic approach combining protein engineering and pathway compartmentation was exploited in Yarrowia lipolytica for the high-efficient production of trans-nerolidol, a sesquiterpene with various commercial applications. Through the single-gene overexpression, the reaction catalyzed by nerolidol synthase (FaNES1) was identified as another rate-limiting step. An optimized FaNES1G498Q was then designed by rational protein engineering using homology modeling and docking studies. Additionally, further improvement of trans-nerolidol production was observed as enhancing the expression of an endogenous carnitine acetyltransferase (CAT2) putatively responsible for acetyl-CoA shuttling between peroxisome and cytosol. To harness the peroxisomal acetyl-CoA pool, a parallel peroxisomal pathway starting with acetyl-CoA to trans-nerolidol was engineered. Finally, the highest reported titer of 11.1 g/L trans-nerolidol in the Y. lipolytica platform was achieved in 5 L fed-batch fermentation with the carbon restriction approach.

De novo biosynthesis of 8-prenylnaringenin in Saccharomyces cerevisiae improved by screening and engineering of prenyltransferases and precursor pathway

8-Prenylnaringenin (8-PN) is a valuable medical phytoestrogen, which is a precursor to many prenylated flavonoids. However, the availability of 8-PN is limited by inefficient prenyltransferases (PTs) and inadequate substrate precursor levels in microbial chassis. First, six PTs from different sources and their truncated cognates were expressed in a (2S)-naringenin producing strain. Only SfN8DT-1 derived from Sophora flavescens and its truncated cognate, tSfN8DT-1, could synthesize 8-PN. Second, tSfN8DT-1 was engineered by multiple sequence alignment and a mutant tSfN8DT-1\(^{Q12E}\) with greater catalytic activity was obtained. Third, key genes, tHMGR and IDI1, of the mevalonate (MVA) pathway were overexpressed using a copy number combinatorial strategy, which greatly improved 8-PN titer by 368.75%. Fourth, a predicted structure of tSfN8DT-1\(^{Q12E}\) was used for molecular docking and virtual saturation mutagenesis. Two key residues, P229 and N305, were identified and saturation mutagenesis on these two sites resulted in an improved mutant N305M. The best-performing mutant, tSfN8DT-1\(^{Q12E N305M}\), produced 49.35 \(\pm {}\) 0.05 mg/L (5.57 \(\pm {}\) 0.01 mg/g DCW) 8-PN in a shaking flask. Finally, 101.40 \(\pm {}\) 2.55 mg/L of 8-PN was obtained in a 5-L bioreactor, which is the greatest titer reported to date for 8-PN. This study combined metabolic engineering and protein engineering methods to enhance precursor supplements and improve the catalytic ability of SfN8DT-1. The production of 8-PN in Saccharomyces cerevisiae was greatly increased through these methods, which may provide a feasible strategy for the biosynthesis of prenylated flavonoids.

Protein Engineering and HDX Identify Structural Regions of G-CSF Critical to Its Stability and Aggregation.

The protein engineering and formulation of therapeutic proteins for prolonged shelf-life remain a major challenge in the biopharmaceutical industry. Understanding the influence of mutations and formulations on the protein structure and dynamics could lead to more predictive approaches to their improvement. Previous intrinsic fluorescence analysis of the chemically denatured granulocyte colony-stimulating factor (G-CSF) suggested that loop AB could subtly reorganize to form an aggregation-prone intermediate state. Hydrogen deuterium exchange mass spectrometry (HDX-MS) has also revealed that excipient binding increased the thermal unfolding transition midpoint (Tm) by stabilizing loop AB. Here, we have combined protein engineering with biophysical analyses and HDX-MS to reveal that increased exchange in a core region of the G-CSF comprising loop AB (ABI, a small helix, ABII) and loop CD packed onto helix B and the beginning of loop BC leads to a decrease in Tm and higher aggregation rates. Furthermore, some mutations can increase the population of the aggregation-prone conformation within the native ensemble, as measured by the greater local exchange within this core region.

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and invitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

Interactions between nascent proteins and the ribosome surface inhibit co-translational folding

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome.

Engineering multifunctional metal/protein hybrid nanomaterials as tools for therapeutic intervention and high-sensitivity detection.

Protein-based hybrid nanomaterials have recently emerged as promising platforms to fabricate tailored multifunctional biologics for biotechnological and biomedical applications. This work shows a simple, modular, and versatile strategy to design custom protein hybrid nanomaterials. This approach combines for the first time the engineering of a therapeutic protein module with the engineering of a nanomaterial-stabilizing module within the same molecule, resulting in a multifunctional hybrid nanocomposite unachievable through conventional material synthesis methodologies. As the first proof of concept, a multifunctional system was designed ad hoc for the therapeutic intervention and monitoring of myocardial fibrosis. This hybrid nanomaterial combines a designed Hsp90 inhibitory domain and a metal nanocluster stabilizing module resulting in a biologic drug labelled with a metal nanocluster. The engineered nanomaterial actively reduced myocardial fibrosis and heart hypertrophy in an animal model of cardiac remodeling. In addition to the therapeutic effect, the metal nanocluster allowed for in vitro, ex vivo, and in vivo detection and imaging of the fibrotic disease under study. This study evidences the potential of combining protein engineering and protein-directed nanomaterial engineering approaches to design custom nanomaterials as theranostic tools, opening up unexplored routes to date for the next generation of advanced nanomaterials in medicine.

Structure and Mechanogating of the Mechanosensitive PIEZO Channel

The evolutionarily conserved PIEZO family of proteins, including PIEZO1 and PIEZO2, forms the long-sought bona fide mammalian mechanosensitive cation channel, and plays critical roles in various mechanotransduction processes such as touch, proprioception, tactile pain, vascular development and blood pressure regulation. Taking a multidisciplinary approach combining protein engineering and purification, cryo-EM, electrophysiology and drug screening, we have determined the three-bladed, propeller-like trimeric structures of both PIEZO1 and PIEZO2 that comprises 114 transmembrane helices (38 per protomer). Transmembrane helices 1-36 (TM1-36) are folded into nine tandem transmembrane helical units (THU) of four TMs each to form the unusual non-planar blades. The three blades are collectively curved into a nano-bowl shape of 28-nm diameter and 10-nm depth, with an extracellular cap-like structure embedded in the centre and a 9-nm-long intracellular beam connecting to the central pore. TM38 and the C-terminal domain are surrounded with the anchor domain and TM37, and enclose the central pore with both transmembrane and cytoplasmic constriction sites. Structural comparison between PIEZO1 and PIEZO2 has revealed that the transmembrane constriction site might act as a transmembrane gate. Structural and functional analyses have suggested that the PIEZO channel might employ the top cap to control the transmembrane gate, while the blade-beam might function as a lever-like apparatus to allosterically control the putative cytosolic gate, indicating a dual-gating mechanism. Together, our studies provide insights into the structure and mechanogating mechanism of PIEZO channels.

Sequence–Structure–Binding Relationships Reveal Adhesion Behavior of the Car9 Solid-Binding Peptide: An Integrated Experimental and Simulation Study

Solid-binding peptides (SBPs) recognizing inorganic and synthetic interfaces have enabled a broad range of materials science applications and hold promise as adhesive or morphogenetic control units that can be genetically encoded within desirable or designed protein frameworks. To date, the underlying relationships governing both SBP-surface and SBP-SBP interactions and how they give rise to different adsorption mechanisms remain unclear. Here, we combine protein engineering, SPR characterization, and molecular dynamics (MD) simulations initiated from Rosetta predictions to gain insights on the interplay of amino acid composition, structure, self-association and adhesion modality in a panel of variants of the Car9 silica-binding peptide (DSARGFKKPGKR) fused to the C-terminus of superfolder green fluorescent protein (sfGFP). Analysis of kinetics, energetics and MD-predicted structures shows that the high affinity binding of Car9 to the silanol-rich surface of silica is dominated by electrostatic contributions and a spectrum of several persistent interactions that, along with a high surface population of bound molecules, promote cooperative interactions between neighboring SBPs and higher order structure formation. Transition from cooperative to Langmuir adhesion in sfGFP-Car9 variants occurs in concert with a reduction of stable surface interactions and self-association, as confirmed by AFM imaging of proteins exhibiting the two different binding behaviors. We discuss the implications of these results for the de novo design of SBP-surface binding systems.

Rigorous Model-Based Design and Experimental Verification of Enzyme-Catalyzed Carboligation under Enzyme Inactivation

Enzyme catalyzed reactions are complex reactions due to the interplay of the enzyme, the reactants, and the operating conditions. To handle this complexity systematically and make use of a design space without technical restrictions, we apply the model based approach of elementary process functions (EPF) for selecting the best process design for enzyme catalysis problems. As a representative case study, we consider the carboligation of propanal and benzaldehyde catalyzed by benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) to produce (R)-2-hydroxy-1-phenylbutan-1-one, because of the substrate dependent reaction rates and the challenging substrate dependent PfBAL inactivation. The apparatus independent EPF concept optimizes the material fluxes influencing the enzyme catalyzed reaction for the given process intensification scenarios. The final product concentration is improved by 13% with the optimized feeding rates, and the optimization results are verified experimentally. In general, the rigorous model driven approach could lead to selecting the best existing reactor, designing novel reactors for enzyme catalysis, and combining protein engineering and process systems engineering concepts.

Minute-scale persistence of a GPCR conformation state triggered by non-cognate G protein interactions primes signaling

Despite the crowded nature of the cellular milieu, ligand–GPCR–G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor–effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR–G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the β2-adrenergic receptor (β2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. β2-AR–Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.

Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in Yarrowia lipolytica.

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.

Mechanical Unfolding and Folding of a Complex Slipknot Protein Probed by Using Optical Tweezers.

Knotted and slipknotted proteins are topologically complex. Understanding their folding and unfolding mechanism has attracted considerable interest. Here we combined protein engineering, single-molecule optical tweezers, and steered molecular dynamics (SMD) simulations to investigate the mechanical unfolding and folding of a slipknotted protein pyruvoyl-dependent arginine decarboxylase (PADC). In its slipknotted structure, PADC contains a long threaded loop (85 residues), which is almost twice the size of the knotting loop. When stretched from its N- and C-termini, the majority of PADC can be readily unfolded in a two-state manner, and the slipknotted structure was untied. A small percentage of PADC unfolded following a three-state pathway involving the formation of an unfolding intermediate state. These unfolding intermediate states showed a broad distribution of contour length increments, suggesting that they did not have a well-defined specific structure. SMD simulations revealed the main free energy barrier to the unfolding of PADC and suggested that the unfolding intermediate states may originate from the frication of polypeptide chain sliding during the process of pulling the threaded loop out of the knotting loop. Upon relaxation, a small percentage of the unfolded and untied PADC polypeptide chain can refold back to its native slipknotted conformation, but a large fraction can only reach a misfolded state. Our results revealed the complexity of the mechanical unfolding and refolding of a slipknotted protein with a long threaded loop.

Study on a 65-mer peptide mimetic enzyme with GPx and SOD dual function.

Excessive reactive oxygen species (ROS) levels are harmful to the body. The peroxidase, GPx, and the superoxide dismutase, SOD, are important antioxidant enzymes for preventing ROS-induced damage. Se-CuZn-65P is an enzyme mimetic with dual GPx and SOD antioxidant function. However, currently, its production is mainly based on the cysteine auxotrophic expression technique, which is inefficient, expensive, and time consuming. In this study, we combined protein engineering and the chemical mutation method to synthesize Se-CuZn-65P. The DNA sequence encoding the 65 amino acid peptide with the desired sequence transformations to incorporate the SOD and the GPx catalytic sites was cloned and expressed in a soluble protein expression vector. The protein yield increased up to 152mg/L, which is 10 times higher than in previous studies. The SOD and GPx activity of Se-CuZn-65P was high (1181U/mg and 753U/μmol, respectively). The binding constant of glutathione was 5.6×104 L·mol-1 , which shows that Se-CuZn-65P efficiently catalyzed hydrogen peroxide reduction by glutathione. Mitochondrial damage experiments confirmed the double protective role of the Se-CuZn-65P peptide and demonstrated functional synergy between the SOD and the GPx domains, which indicates its potential to be used in the treatment of ROS-related diseases. Our research may give a new thought to increase the yield of mimic.

Spatiotemporal imaging of small GTPases activity in live cells

Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Förster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.