Conformational Changes Research Articles

1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA’s depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.

Interaction of exogenous acetylcholinesterase and butyrylcholinesterase with amyloid-β plaques in human brain tissue

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are associated with amyloid-β (Aβ) plaques and exhibit altered biochemical properties in human Alzheimer's disease (AD), as well as in the transgenic 5XFAD mouse model of AD amyloidosis. In the brains of the 5XFAD mouse model devoid of BChE enzyme (5XFAD/BChE-KO), incubation of tissue sections with exogenous BChE purified from human plasma (pl-BChE) leads to its association with Aβ plaques and its biochemical properties are comparable to those reported for endogenous BChE associated with plaques in both human AD and in 5XFAD mouse brain tissue. We sought to determine whether these observations in 5XFAD/BChE-KO mice also apply to human brain tissues. To do so, endogenous ChE activity in human AD brain tissue sections was quenched with 50 % aqueous acetonitrile (MeCNaq) leaving the tissue suitable for further studies. Quenched sections were then incubated with recombinant AChE (r-AChE) or pl-BChE and stained for each enzymes' activity. Exogenous r-AChE or pl-BChE became associated with Aβ plaques, and when bound, had properties that were comparable to the endogenous ChE enzymes associated with plaques in AD brain tissues without acetonitrile treatment. These findings in human AD brain tissue extend previous observations in the 5XFAD/BChE-KO mouse model and demonstrate that exogenously applied r-AChE and pl-BChE have high affinity for Aβ plaques in human brain tissues. This association alters the biochemical properties of these enzymes, most likely due a conformational change. If incorporation of AChE and BChE in Aβ plaques facilitates AD pathogenesis, blocking this association could lead to disease-modifying approaches to AD. This work provides a method to study the mechanism of AChE and BChE interaction with Aβ plaque pathology in post-mortem human brain tissue.

Solution NMR chemical shift assignment of apo and molybdate-bound ModA at two pHs.

ModA is a soluble periplasmic molybdate-binding protein found in most gram-negative bacteria. It is part of the ABC transporter complex ModABC that moves molybdenum into the cytoplasm, to be used by enzymes that carry out various redox reactions. Since there is no clear analog for ModA in humans, this protein could be a good target for antibacterial drug design. Backbone 1H, 13C and 15N chemical shifts of apo and molybdate-bound ModA from E. coli were assigned at pHs 6.0 and 4.5. In addition, side chain atoms were assigned for apo ModA at pH 6.0. When comparing apo and molybdate-bound ModA at pH 6.0, large chemical shift perturbations are observed, not only in areas near the bound metal, but also in regions that are distant from the metal-binding site. Given the significant conformational change between apo and holo ModA, we might expect the large chemical shift changes to be more widespread; however, since they are limited to specific regions, the residues with large perturbations may reveal allosteric sites that could ultimately be important for the design of antibiotics that target ModA.

Analysis of the effect of cations on protein conformational stability using solid-state nanopores.

The conformation of proteins is closely related to their biological functions, and it is affected by many factors, including the type of cations in solution. However, it is difficult to detect the conformational changes of a protein in situ. As a single-molecule sensing technology, nanopores can convert molecular structural information into analyzable current signals within a reasonable time range. Herein, we detect and analyze the effects of two different types of monovalent cations (Na+ and Li+) on a model protein bovine serum albumin (BSA) conformation using SiNx nanopores with different diameters. The quantitative analysis results show that the excluded volume of BSA in LiCl salt solutions is larger than the value in NaCl solution, indicating that Li+ is more prone to unfolding the proteins and making them unstable. This study demonstrated that nanopores enable the in situ detection of the structure of proteins at the single-molecule level and provide a new approach for the quantitative analysis of proteins.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

Conformational Modulation of Tissue Transglutaminase via Active Site Thiol Alkylating Agents: Size Does Not Matter.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.

Clutch model for focal adhesions predicts reduced self-stabilization under oblique pulling

Cell-matrix adhesions connect the cytoskeleton to the extracellular environment and are essential for maintaining the integrity of tissue and whole organisms. Remarkably, cell adhesions can adapt their size and composition to an applied force such that their size and strength increases proportionally to the load. Mathematical models for the clutch-like force transmission at adhesions are frequently based on the assumption that mechanical load is applied tangentially to the adhesion plane. Recently, we suggested a molecular mechanism that can explain adhesion growth under load for planar cell adhesions. The mechanism is based on conformation changes of adhesion molecules that are dynamically exchanged with a reservoir. Tangential loading drives the occupation of some states out of equilibrium, which for thermodynamic reasons, leads to the association of further molecules with the cluster, which we refer to as self-stabilization. Here, we generalize this model to forces that pull at an oblique angle to the plane supporting the cell, and examine if this idealized model also predicts self-stabilization. We also allow for a variable distance between the parallel planes representing cytoskeletal F-actin and transmembrane integrins. Simulation results demonstrate that the binding mechanism and the geometry of the cluster have a strong influence on the response of adhesion clusters to force. For oblique angles smaller than about 40∘, we observe a growth of the adhesion site under force. However this self-stabilization is reduced as the angle between the force and substrate plane increases, with vanishing self-stabilization for normal pulling. Overall, these results highlight the fundamental difference between the assumption of pulling and shearing forces in commonly used models of cell adhesion.

Engineering Amino Acid and Peptide Supramolecular Architectures through Fluorination.

Fluorinated non-natural amino acids are attracting considerable research interest, especially in the biomedical field and in materials science, thanks to their ability to self-assemble into peculiar supramolecular structures. The conformational changes induced by the presence of fluorine atoms obviously affect their functions, as well as the biological activity of the deriving peptides and proteins. Here, we will briefly describe the main effects of fluorination on the aggregation behavior of such building blocks, focusing in particular on their improved tendency to form fibrils, and gels therefrom. Our aim is to underline the promising potential of fluorination as a tool to affect the self-assembly features of amino acids, both when used alone and when inserted into polypeptide sequences. The ability of fluorine to influence physical, chemical, and structural properties of these substrates offers the possibility to engineer bioinspired materials with specific and tunable functions.

N-Methyl-D-Aspartate (NMDA) Receptor Antagonists and Their Pharmacological Implication: A Medicinal Chemistry-Oriented Perspective Outline.

N-methyl-D-aspartate (NMDA) receptors, i.e., inotropic glutamate receptors, are important in synaptic plasticity, brain growth, memory, and learning. The activation of NMDA is done by neurotransmitter glutamate and co-agonist (glycine or D-serine) binding. However, the over-activation of NMDA elevates the intracellular calcium influx, which causes various neurological diseases and disorders. Therefore, to prevent excitotoxicity and neuronal death, inhibition of NMDA must be done using its antagonist. This review delineates the structure of subunits of NMDA and the conformational changes induced after the binding of agonists (glycine and D-serine) and antagonists (ifenprodil, etc.). Additionally, reported NMDA antagonists from different sources, such as synthetic, semisynthetic, and natural resources, are explained by their mechanism of action and pharmacological role. The comprehensive report also addresses the chemical spacing of NMDA inhibitors and in-vivo and in-vitro models to test NMDA antagonists. Since the Blood-Brain Barrier (BBB) is the primary membrane that prevents the penetration of a wide variety of drug molecules, we also elaborate on the medicinal chemistry approach to improve the effectiveness of their antagonists.

Mechanical Stability and Unfolding Pathways of Parallel Tetrameric G-Quadruplexes Probed by Pulling Simulations.

Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects remain elusive, despite being important for understanding structure formation and biological functions of GQs. Pulling experiments are a common tool for acquiring insights into the folding landscape of GQs. Herein, we applied a computational pulling strategy─steered molecular dynamics (SMD) simulations─in combination with standard molecular dynamics (MD) simulations to explore the unfolding landscapes of tetrameric parallel GQs. We identified anisotropic properties of elastic conformational changes, unfolding transitions, and GQ mechanical stabilities. Using a special set of structural parameters, we found that the vertical component of pulling force (perpendicular to the average G-quartet plane) plays a significant role in disrupting GQ structures and weakening their mechanical stabilities. We demonstrated that the magnitude of the vertical force component depends on the pulling anchor positions and the number of G-quartets. Typical unfolding transitions for tetrameric parallel GQs involve base unzipping, opening of the G-stem, strand slippage, and rotation to cross-like structures. The unzipping was detected as the first and dominant unfolding event, and it usually started at the 3'-end. Furthermore, results from both SMD and standard MD simulations indicate that partial spiral conformations serve as a transient ensemble during the (un)folding of GQs.

Stepwise activation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors belong to a family of Gprotein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the Gprotein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and Gprotein binding.

In silico study of alkaloids with quercetin nucleus for inhibition of SARS-CoV-2 protease and receptor cell protease.

Covid-19 disease caused by the deadly SARS-CoV-2 virus is a serious and threatening global health issue declared by the WHO as an epidemic. Researchers are studying the design and discovery of drugs to inhibit the SARS-CoV-2 virus due to its high mortality rate. The main Covid-19 virus protease (Mpro) and human transmembrane protease, serine 2 (TMPRSS2) are attractive targets for the study of antiviral drugs against SARS-2 coronavirus. Increasing consumption of herbal medicines in the community and a serious approach to these drugs have increased the demand for effective herbal substances. Alkaloids are one of the most important active ingredients in medicinal plants that have wide applications in the pharmaceutical industry. In this study, seven alkaloid ligands with Quercetin nucleus for the inhibition of Mpro and TMPRSS2 were studied using computational drug design including molecular docking and molecular dynamics simulation (MD). Auto Dock software was used to evaluate molecular binding energy. Three ligands with the most negative docking score were selected to be entered into the MD simulation procedure. To evaluate the protein conformational changes induced by tested ligands and calculate the binding energy between the ligands and target proteins, GROMACS software based on AMBER03 force field was used. The MD results showed that Phyllospadine and Dracocephin-A form stable complexes with Mpro and TMPRSS2. Prolinalin-A indicated an acceptable inhibitory effect on Mpro, whereas it resulted in some structural instability of TMPRSS2. The total binding energies between three ligands, Prolinalin-A, Phyllospadine and Dracocephin-A and two proteins MPro and TMRPSS2 are (-111.235 ± 15.877, - 75.422 ± 11.140), (-107.033 ± 9.072, -84.939 ± 10.155) and (-102.941 ± 9.477, - 92.451 ± 10.539), respectively. Since the binding energies are at a minimum, this indicates confirmation of the proper binding of the ligands to the proteins. Regardless of some Prolinalin-A-induced TMPRSS2 conformational changes, it may properly bind to TMPRSS2 binding site due to its acceptable binding energy. Therefore, these three ligands can be promising candidates for the development of drugs to treat infections caused by the SARS-CoV-2 virus.

Design and synthesis of peptides as stabilizers of histone deacetylase 4.

Histone deacetylase 4 (HDAC4) contributes to gene repression by complex formation with HDAC3 and the corepressor silencing mediator for retinoid or thyroid hormone receptors (SMRT). We hypothesized that peptides derived from the class IIa specific binding site of SMRT would stabilize a specific conformation of its target protein and modulate its activity. Based on the SMRT-motif 1 (SM1) involved in the interaction of SMRT with HDAC4, we systematically developed cyclic peptides that exhibit Ki values that are 9 to 56 times lower than that of the linear SMRT peptide. The peptide macrocycles stabilize the wildtype of the catalytic domain of HDAC4 (cHDAC4) considerably better than its thermally more stable 'gain-of-function' (GOF) variant, cHDAC4-H976Y. Molecular docking and mutagenesis studies indicated that the cyclic peptides bind in a similar but not identical manner as the linear SMRT peptide to a discontinuous binding site. Ion mobility mass spectrometry showed no major changes in the protein fold upon peptide binding. Consistent with these results, preliminary hydrogen-deuterium exchange mass spectrometry measurements indicated only minor conformational changes. Taken together, the cyclic SMRT peptides most likely stabilize the apo form of cHDAC4.

Monitoring of DNA structural changes after incorporation of the phenylpyrazole insecticide fipronil

The use of insecticides presents a risk to the environment because they can accumulate in the water, soil, air, and organisms, endangering human and animal health. It is therefore essential to investigate the effects of different groups of insecticides on individual biomacromolecules such as DNA. We studied fipronil, which belongs to the group of phenylpyrazole insecticides. The interaction of fipronil with calf thymus DNA was investigated using spectroscopic methods (absorption and fluorescence spectroscopy) complemented with infrared spectroscopy and viscosity measurement. Fluorescence emission spectroscopy showed the formation of a fipronil/DNA complex with a combined static and dynamic type of quenching. The binding constant was 4.15 × 103 L/mol. Viscosity changes were recorded to confirm/disconfirm the intercalation mode of interaction. A slight change in DNA viscosity in the presence of fipronil was observed. The phenylpyrazole insecticide does not cause significant conformational changes in DNA structure or increase of its chain length. We hypothesize that fipronil is incorporated into the minor groove of the DNA macromolecule via hydrogen interactions as indicated by FT-IR and CD measurements.

Anion‐Coordination Foldamer‐Based Polymer Network: from Molecular Spring to Elastomer

Foldamer is a scaled‐down version of coil spring, which can absorb and release energy by conformational change. Here, polymer networks with high‐density of molecular springs were developed by employing anion‐coordination‐based foldamers as the monomer. The coiling of the foldamer is controlled by oligourea ligands coordinating to chloride ions; subsequently, the folding and unfolding of foldamer conformations endow the polymer network with excellent energy dissipation and toughness. The mechanical performance of the corresponding polymer network shows a dramatic increase from P‐L2UCl (non‐folding), P‐L4UCl (a full turn) to P‐L6UCl (1.5 turns), in terms of strength (2.62 MPa; 14.26 Mpa; 22.93 Mpa), elongation at break (70%; 325%; 352%), Young’s modulus (2.69 MPa; 63.61 Mpa; 141.50 Mpa), and toughness (1.12 MJ/m3; 21.39 MJ/m3; 49.62 MJ/m3), respectively, which are also better than those without anion centers and the non‐foldamer based counterparts. Moreover, P‐L6UCl shows enhanced strength and toughness than most of the molecular‐spring based polymer networks.Moreover, P‐L6UCl shows enhanced strength and toughness than most of the molecular‐spring based polymer networks. Thus, an effective strategy for designing high‐performance anion‐coordination‐based materials is presented in this study.

Glycerophospholipid polyunsaturation modulates resveratrol action on biomimetic membranes

The phytoalexin resveratrol has received increasing attention for its potential to prevent oxidative damages in human organism. To shed further light on molecular mechanisms of its interaction with lipid membranes we study resveratrol influence on the organisation and mechanical properties of biomimetic lipid systems composed of synthetic phosphatidylcholines with mixed aliphatic chains and different degree of unsaturation at sn-2 position (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, PDPC). High-sensitivity isothermal titration calorimetric measurements reveal stronger spontaneous resveratrol association to polyunsaturated phosphatidylcholine bilayers compared to the monounsaturated ones resulting from hydrophobic interactions, conformational changes of the interacting species and desolvation of molecular surfaces. The latter is supported by the results from Laurdan spectroscopy of large unilamellar vesicles providing data on hydration at the glycerol backbones of glycerophospholipides. Higher degree of lipid order is reported for POPC membranes compared to PDPC. While resveratrol mostly enhances the hydration of PDPC membranes, increasing POPC dehydration is reported upon treatment with the polyphenol. Dehydration of the polyunsaturated lipid bilayers is measured only at the highest phytoalexin content studied (resveratrol/lipid 0.5 mol/mol) and is less pronounced than the effect reported for POPC membranes. The polyphenol effect on membrane mechanics is probed by thermal shape fluctuation analysis of quasispherical giant unilamellar vesicles. Markedly different trend of the bending elasticity with increasing resveratrol concentration is reported for the two types of phospholipid bilayers studied. POPC membranes become more rigid in the presence of resveratrol, whereas PDPC-containing bilayers exhibit softening at lower concentrations of the polyphenol followed by a slight growth without bilayer stiffening even at the highest resveratrol content explored. The new data on the structural organization and membrane properties of resveratrol-treated phosphatidylcholine membranes may underpin the development of future liposomal applications of the polyphenol in medicinal chemistry.

Anion-Coordination Foldamer-Based Polymer Network: from Molecular Spring to Elastomer.

Foldamer is a scaled-down version of coil spring, which can absorb and release energy by conformational change. Here, polymer networks with high-density of molecular springs were developed by employing anion-coordination-based foldamers as the monomer. The coiling of the foldamer is controlled by oligourea ligands coordinating to chloride ions; subsequently, the folding and unfolding of foldamer conformations endow the polymer network with excellent energy dissipation and toughness. The mechanical performance of the corresponding polymer network shows a dramatic increase from P-L2UCl (non-folding), P-L4UCl (a full turn) to P-L6UCl (1.5 turns), in terms of strength (2.62 MPa; 14.26 Mpa; 22.93 Mpa), elongation at break (70%; 325%; 352%), Young's modulus (2.69 MPa; 63.61 Mpa; 141.50 Mpa), and toughness (1.12 MJ/m3; 21.39 MJ/m3; 49.62 MJ/m3), respectively, which are also better than those without anion centers and the non-foldamer based counterparts.Moreover, P-L6UCl shows enhanced strength and toughness than most of the molecular-spring based polymer networks.Moreover, P-L6UCl shows enhanced strength and toughness than most of the molecular-spring based polymer networks. Thus, an effective strategy for designing high-performance anion-coordination-based materials is presented in this study.

Understanding the Modulatory Role of E2012 on the γ-Secretase-Substrate Interaction.

Allosteric modulation plays a critical role in enzyme functionality and requires a deep understanding of the interactions between the active and allosteric sites. γ-Secretase (GS) is a key therapeutic target in the treatment of Alzheimer's disease (AD), through its role in the synthesis of amyloid β peptides that accumulate in AD patients. This study explores the structure and dynamic effects of GS modulation by E2012 binding, employing well-tempered metadynamics and conventional molecular dynamics simulations across three binding scenarios: (1) GS enzyme with and without L458 inhibitor, (2) the GS-substrate complex together with the modulator E2012 in two different binding modes, and (3) E2012 interacting with a C99 substrate fragment. Our findings reveal that the presence of L458 induces conformational changes that contribute to stabilization of the GS enzyme dynamics, previously reported as a key factor that allowed the resolution of the cryo-EM structure and the enhanced binding of E2012. Furthermore, we identified the most favorable binding site for E2012 within the GS-substrate complex, uncovering significant modulatory effects and a complex network of interactions that influence the position of the substrate for catalysis. In addition, we explore a potential substrate-modulator binding before the formation of the enzyme-substrate complex. The insights gained from our study emphasize the importance of these interactions in the development of potential therapeutic interventions that target the functionality of the GS enzyme in AD.

Self-Assembled Recombinant Elastin and Globular Protein Vesicles with Tunable Properties for Diverse Applications.

ConspectusVesicles are self-assembled structures comprised of a membrane-like exterior surrounding a hollow lumen with applications in drug delivery, artificial cells, and micro-bioreactors. Lipid or polymer vesicles are the most common and are made of lipids or polymers, respectively. They are highly useful structures for many applications but it can be challenging to decorate them with proteins or encapsulate proteins in them, owing to the use of organic solvent in their formation and the large size of proteins relative to lipid or polymer molecules. By utilization of recombinant fusion proteins to make vesicles, specific protein domains can be directly incorporated while also imparting tunability and stability. Protein vesicle assembly relies on the design and use of self-assembling amphiphilic proteins. A specific protein vesicle platform made in purely aqueous conditions of a globular, functional protein fused to a glutamate-rich leucine zipper (ZE) and a thermoresponsive elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) is discussed here. The hydrophobic conformational change of the ELP above its transition temperature drives assembly, and strong ZE/ZR binding enables incorporation of the desired functional protein. Mixing the soluble proteins on ice induces zipper binding, and then warming above the ELP transition temperature (Tt) triggers the transition to and growth of protein-rich coacervates and, finally, reorganization of proteins into vesicles. Vesicle size is tunable based on salt concentration, rate of heating, protein concentration, size of the globular protein, molar ratio of the proteins, and the ELP sequence. Increasing the salt concentration decreases vesicle size by decreasing the Tt, resulting in a shorter coacervation transition stage. Likewise, directly changing the heating rate also changes this time and increasing protein concentration increases coalescence. Increasing globular protein size decreases the size of the vesicle due to steric hindrance. By changing the ELP sequence, which consists of (VPGXG)n, through the guest residue (X) or number of repeats (n), Tt is changed, affecting size. Additionally, the chemical nature of X variation has endowed vesicles with stimuli responsiveness and stability at physiological conditions.Protein vesicles have been used for biocatalysis, biomacromolecular drug delivery, and vaccine applications. Photo-cross-linkable vesicles were used to deliver small molecule cargo to cancer cells in vitro and antigen to immune cells in vivo. pH-responsive vesicles effectively delivered functional protein cargo, including cytochrome C, to the cytosol of cancer cells in vitro, using hydrophobic ion pairing to improve cargo distribution in the vesicles and release. The globular protein used to make the vesicles can be varied to achieve different functions. For example, enzyme vesicles exhibit biocatalysis, and antigen vesicles induce antibody and cellular immune responses after vaccination in mice. Collectively, the development and engineering of the protein vesicle platform has employed amphiphilic self-assembly strategies and rational protein engineering to control physical, chemical, and biological properties for biotechnology and nanomedicine applications.

Spatiotemporal Control of Protein Refolding through Flash-Change Reaction Conditions.

Recombinant protein production is an essential aspect of biopharmaceutical manufacturing, with Escherichia coli serving as a primary host organism. Protein refolding is vital for protein production; however, conventional refolding methods face challenges such as scale-up limitations and difficulties in controlling protein conformational changes on a millisecond scale. In this study, we demonstrate the novel application of flow microreactors (FMR) in controlling protein conformational changes on a millisecond scale, enabling efficient refolding processes and opening up new avenues in the science of FMR technology. FMR technology has been primarily employed for small-molecule synthesis, but our novel approach successfully expands its application to protein refolding, offering precise control of the buffer pH and solvent content. Using interleukin-6 as a model, the system yielded an impressive 96% pure refolded protein and allowed for gram-scale production. This FMR system allows flash changes in the reaction conditions, effectively circumventing protein aggregation during refolding. To the best of our knowledge, this is the first study to use FMR for protein refolding, which offers a more efficient and scalable method for protein production. The study results highlight the utility of the FMR as a high-throughput screening tool for streamlined scale-up and emphasize the importance of understanding and controlling intermediates in the refolding process. The FMR technique offers a promising approach for enhancing protein refolding efficiency and has demonstrated its potential in streamlining the process from laboratory-scale research to industrial-scale production, making it a game-changing technology in the field.