Search

Ferritin is a 12 nanometer (nm) diameter iron storage protein complex that is found in most plants and animals. A substantial body of evidence has established that electrons can tunnel through and between ferritin protein nanoparticles and that it exhibits Coulomb blockade behavior, similar to quantum dots and nanoparticles. This evidence can be used to understand the behavior of these particles for use in nanoelectronic devices, for biomedical applications and for investigation of quantum biological phenomena. Ferritin also has magnetic properties that make it useful for applications such as memristors and as a contrast agent for magnetic resonance imaging. This article provides a short overview of this evidence, as well as evidence of ferritin structures in vivo and of tunneling in those structures, with an emphasis on ferritin structures in substantia nigra pars compacta (SNc) neurons. Potential biomedical applications that could utilize these ferritin protein nanoparticles are also discussed.

Plastic pollution in diverse terrestrial and marine environments is a widely recognised and growing problem. Bio-recycling and upcycling of plastic waste is a potential solution to plastic pollution, as these processes convert plastic waste into useful materials. Polyethylene terephthalate (PET) is the most abundant plastic waste, and this material can be degraded by a class of recently discovered bacterial esterase enzymes known as PET hydrolases (PETase). Investigations of the enzymatic hydrolysis of diverse PET molecules have clearly revealed that the biodegradability of various PET substrates depends on both their chemical structure and physical properties, including polymer length, crystallinity, glass transition temperature, surface area, and surface charge. This review summarises the known impacts of crystallinity and other physical properties on enzymatic PET hydrolysis. National Research Foundation (NRF) Published version This publication is supported by the National Research Foundation, Singapore, under its Competitive Research Programme (Award# NRF-CRP22-2019-0005)

Bacterial cellulose (BC) has been utilized as a biopolymer matrix for various applications. The advancement of synthetic biology has brought new approaches for its production and functionalization. In this mini-review, we briefly discuss the conventional methods to improve BC production and functionalization, and their challenges. We summarize the application of synthetic biology to address these challenges and its use to develop novel hybrid living materials. Finally, we consider the opportunities and future prospects of synthetic biology in bioengineered materials. National Research Foundation (NRF) This research was partially funded by the Asian Office of Aerospace Research and Development (AOARD) Grant (#FA2386-19-1-4060) and National Research Foundation, Singapore, under its Competitive Research Programme (Award#NRF-CRP22-2019-0005).

Nanoparticles (NPs), both organic and inorganic, have been identified as tools for diagnostic and therapeutic (theranostic) applications. Macrophages constitute the first line of defense in the human body following the introduction of foreign antigens, including nanoparticles. However, there is a limited understanding of the cellular fate and trafficking of organic NPs in macrophages as well as the molecular responses that are triggered. This knowledge is crucial for the effective translation of these engineered molecules for theranostic applications. In this work, we performed an in-depth study on the intracellular fate and relevant immune responses of a model organic NP, Archaeoglobus fulgidus ferritin, in murine macrophage (RAW264.7) cells. Ferritin, a naturally occurring iron storage protein, has been reported to target tumors and atherosclerotic lesion sites. Herein, we demonstrate a concentration-dependent internalization mechanism and quantify the subcellular localization of ferritin NPs in various organelles. After NP exposure, export of the iron present in the ferritin core occurred over an extended period of time along with upregulation of iron-related gene mRNA expression. A study on the modulation of the intracellular localization of the NPs was conducted by incorporating peptides to mediate endosomal escape and examining their molecular effects using transcriptional analysis. To further investigate the physiological effects, we monitored the upregulation of immune-related markers (i.e., CCR2, IL1β, TNFα, VCAM-1) along with ROS generation in cells treated with ferritin under various conditions. The in-depth analyses of cellular uptake and responses to versatile protein NPs, such as ferritin, provide basic principles to design and engineer other protein NPs with similar properties for future biomedical applications. Ministry of Education (MOE) Nanyang Technological University Published version This work is supported in parts by Nanyang Technological University, Singapore Start-Up Grant through the School of Materials Science and Engineering and Institute for Digital Molecular Analytics and Science (IDMxS) at Nanyang Technological University, Singapore through Singapore Ministry of Education funding under the Research Centres of Excellence scheme.

Carbene-based macromolecules are an emerging new stimuli-sensitive class of biomaterials that avoid the impediments of free radical polymerization but maintain a rapid liquid-to-biorubber transition. Activation of diazirine-grafted polycaprolactone polyol (CaproGlu) is limited to UVA wavelengths that have tissue exposure constraints and limited light intensities. For the first time, UVA is circumvented with visible light-emitting diodes at 445 nm (blue) to rapidly activate diazirine-to-carbene covalent cross-linking. Iridium photocatalysts serve to initiate diazirine, despite having little to no absorption at 445 nm. CaproGlu's liquid organic matrix dissolves the photocatalyst with no solvents required, creating a light transparent matrix. Considerable differences in cross-linking chemistry are observed in UVA vs visible/photocatalyst formulations. Empirical analysis and theoretical calculations reveal a more efficient conversion of diazirine directly to carbene with no diazoalkane intermediate detected. Photorheometry results demonstrate a correlation between shear moduli, joules light dose, and the lower limits of photocatalyst concentration required for the liquid-to-biorubber transition. Adhesion strength on ex vivo hydrated tissues exceeds that of cyanoacrylates, with a fixation strength of up to 20 kg·f·cm2. Preliminary toxicity assessment on leachates and materials directly in contact with mammalian fibroblast cells displays no signs of fibroblast cytotoxicity. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University Accepted version T.W.J.S. and I.D. are co-inventors of patent application Hygroscopic, Crosslinking Coatings and Bioadhesives; PCT/ SG2018/050452. The project was supported by the Ministry of Education Tier 1 Grant RG17/18 (S): Novel light activated, diazo protecting groups; Ministry of Education Tier 2 Grant (MOE2018-T2-2-114): CaproGlu, double-sided wet tissue adhesives; NTUitive POC (Gap) Fund NGF/2018/05: Aesthetic Applications of CaproGlu Bioadhesives; and A*Star IAF PP Grant (H19/01/a0/0II9): CathoGlu Bioadhesives-preventing catheter extravasation and skin infections. L.B. acknowledges funding from the Spanish Ministry of Science and Innovation (MCIU), project PID2019- 104654GB-I00.

Coacervates are dense microdroplets formed by liquid-liquid phase separation (LLPS) of macromolecules that have gained increasing attention as drug delivery vehicles. Recently, we have reported a new intracellular delivery system based on self-coacervating histidine (His)-rich beak peptides (HBpep and HBpep-SP) inspired by beak proteins of the Humboldt squid. These peptide microdroplets combine excellent encapsulation efficiency of therapeutics with high transfection rate and low cytotoxicity. However, the mechanism by which they cross the cell membrane remains elusive. Previous inhibitor studies provided incomplete clues into the detail uptake pathway, although they suggested a cholesterol-dependent and, possibly, an energy-independent non-classical mechanism of internalization. In this study, we improved our understanding of coacervates/cell membrane interactions using model membranes, namely Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs). We also employ a combination of electron microscopy techniques to gain detailed structural insights into the cell uptake of HBpep and HBpep-SP coacervates. We demonstrate that modulating lipid charge and cholesterol level influence coacervate attachment to GUVs. However, they are not able to cross the GUV’s lumen in an energy-independent manner. We then show that the coacervates enter HeLa and HepG2 cells via a mechanism sharing morphological features of macropinocytosis and phagocytosis, in particular involving cytoskeleton rearrangement and capture by filipodia. Our study provides key insights into the interaction of HPpep and HBpep-SP coacervates with model membranes as well as their cellular uptake pathway.

The wet environment in the oral cavity is challenging for topical disease management approaches. The compromised material properties leading to weak adhesion and short retention (8 h) in such environment result in frequent reapplication of the therapeutics. Composites of bacterial cellulose (BC) and carbene-based bioadhesives attempt to address these shortcomings. Previous designs comprised of aqueous formulations. The current design, for the first time, presents dry, shelf-stable cellulose patches for convenient ready-to-use application. The dry patches simultaneously remove tissue surface hydration while retaining carbene-based photocuring and offers on-demand adhesion. The dry patch prototypes are optimized by controlling BC/adhesive mole ratios and dehydration technique. The adhesion strength is higher than commercial denture adhesives on soft mucosal tissues. The structural integrity is maintained for a minimum of 7 days in aqueous environment. The patches act as selective nanoporous barrier against bacteria while allowing permeation of proteins. The results support the application of BC-based adhesive patches as a flexible platform for wound dressings, drug depots, or combination thereof.

Bacterial cellulose (BC) based hybrid hydrogels and aerogels have been fabricated by reformatting BC pellicles. BC pellicles are formed by tight fibrillar networks making it challenging to incorporate other materials to form hybrids or composites. In this study, BC pellicles produced from two Acetobacteracea family members are disintegrated into individual nanofibrils via TEMPO-mediated oxidation. The individualized BC nanofibrils about tens of microns in length and 20–50 nm in width are well-dispersed in water with crystallinity and birefringence of cellulose I. These BC nanofibril dispersions are non-Newtonian fluid. Upon complexation with 0.75 mmol/g of Fe3+, the formed BC nanofibril hydrogels display 17-fold increase in storage moduli. Composite aerogels formed by complexation with conductive polymer PEDOT/PSS show characteristics of a non-Hookean foam material with conductivity value of up to 0.86 S/cm for cellulose density of 4.2 mg/cm2.

Enzymes, receptors, and carrier proteins discriminate between enantiomers of natural and synthetic chemicals. Whereas the structural details of this phenomenon have been investigated in enzymes and receptors, much less is known for carrier proteins of hydrophobic ligands, particularly concerning the contribution of asymmetric centers in the side chains of amino acids to chirally selective binding. Working with a pig odorant-binding protein, we have found that the replacement of either one or both isoleucine residues in the binding pocket by leucines abolishes discrimination of menthol and carvone enantiomers. The results indicate that isoleucines are crucial for chiral discrimination of hydrophobic ligands, and that asymmetry in the side chain may be as important as the overall asymmetry of the protein. The results provide suggestions and guidelines for improving chiral selectivity of binding proteins and enzymes, with consequent applications in the production of enantiomerically pure drugs.

The development of protein-polymer complexes using protein cages is garnering attention as a strategy to overcome challenges that relate to immunogenicity and protease degradation. We propose the use of thermoresponsive polymers such as poly(N-isopropylacrylamide) (PNIPAM) to form ferritin-polymer complexes through non-covalent interactions. Ferritin is observed to be released from the complexes after 1–2 days incubation at 37 °C.

Bacterial cellulose (BC), a renewable type of cellulose, has been used in the manufacture of foods, cosmetics, and biomedical products. To produce BC, a high-throughput single-bacterium measurement is necessary to identify the functional bacteria that can produce BC with sufficient amount and desirable morphology. In this study, a continuous-flow intelligent optofluidic device was developed to enable high-throughput single-bacterium profiling of BC. Single bacteria were incubated in agarose hydrogel particles to produce BC with varied densities and structures. An intelligent convolutional neural network (CNN) computational method was developed to analyze the scattering patterns of BC. The BC production and morphology were determined with a throughput of ∼35 bacteria per second. A total of ∼105 single-bacterium BC samples were characterized within 3 hours. The high flexibility of this approach facilitates high-throughput comprehensive single-cell production analysis for a range of applications in engineering biology.

Protein cages (viral and non-viral) found in nature have evolved for a variety of purposes and are found in all kingdoms of life. The main functions of these nanoscale compartments are the protection and delivery of nucleic acids e.g. virus capsids, or the enrichment and sequestration of metabolons e.g. bacterial microcompartments. This review focuses on recent developments of protein cages for use in immunotherapy and therapeutic delivery. In doing so, we highlight the unique ways in which protein cages have informed on fundamental principles governing bio-nano interactions. With the enormous existing design space among naturally occurring protein cages, there is still much to learn from studying them as biomimetic particles.

(1) Background: This paper aims to present and discuss the most significant challenges encountered by STEM professionals associated with remote working during the COVID-19 lockdowns. (2) Methods: We performed a qualitative analysis of 921 responses from professionals from 76 countries to the open-ended question: "What has been most challenging during the lockdown for you, and/or your family?" (3) Findings: Participants reported challenges within the immediate family to include responsibilities for school, childcare, and children's wellbeing; and the loss of social interactions with family and friends. Participants reported increased domestic duties, blurred lines between home and work, and long workdays. Finding adequate workspace was a problem, and adaptations were necessary, especially when adults shared the same setting for working and childcare. Connectivity issues and concentration difficulties emerged. While some participants reported employers' expectations did not change, others revealed concerns about efficiency. Mental health issues were expressed as anxiety and depression symptoms, exhaustion and burnout, and no outlets for stress. Fear of becoming infected with COVID-19 and uncertainties about the future also emerged. Pressure points related to gender, relationship status, and ethnicities were also evaluated. Public policies differed substantially across countries, raising concerns about the adherence to unnecessary restrictions, and similarly, restrictions being not tight enough. Beyond challenges, some benefits emerged, such as increased productivity and less time spent getting ready for work and commuting. Confinement resulted in more quality time and stronger relationships with family. (4) Interpretation: Viewpoints on positive and negative aspects of remote working differed by gender. Females were more affected professionally, socially, and personally than males. Mental stress and the feeling of inadequate work efficiency in women were caused by employers' expectations and lack of flexibility. Working from home turned out to be challenging, primarily due to a lack of preparedness, limited access to a dedicated home-office, and lack of previous experience in multi-layer/multi-scale environments. Published version Funding has been received from the International Union for Physical and Engineering Sciences in Medicine (IUPESM) towards data analysis.

Topical treatments for oral wounds and infections exhibit weak adhesion to wet surfaces which results in short retention duration (6-8 hours), frequent dosing requirement and patient incompatibility. To address these limitations, aqueous composites made of fibrillated bacterial cellulose and photoactive bioadhesives are designed for soft epithelial surfaces. The aqueous composites crosslink upon photocuring within a minute and exhibit a transition from viscous to elastic adhesive hydrogels. The light-cured composites have shear moduli mimicking oral mucosa and other soft tissues. The tunable adhesion strength ranges from 3 to 35 kPa on hydrated tissue-mimicking surfaces (collagen film). The results support the application of bacterial cellulose hydrogel systems for potential treatment of mucosal wounds. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Submitted/Accepted version The project is partially supported by A*STAR IAF PP Grant (H19/01/a0/0II9): CathoGlu Bioadhesives-preventing catheter extravasation and skin infections; Ministry of Education Tier 2 Grant: CaproGlu, Double sided wet-tissue adhesives (MOE2018-T2-2-114).

This IET Engineering Biology special issue in Environment and Sustainability is launched in anticipation of the call for actions to mitigate climate change following the conclusion of the 26th Conference of Parties (COP26) at Glasgow. The mitigation requires multi‐pronged approaches and innovations, that include reduction of coal usage, cutting back methane emission, sequestration of CO2, financing, to name a few. But, more can and need to be done. There is a pressing need to move to environmentally friendly, sustainable low‐carbon so- lutions. Synthetic biology has been mentioned to be one of the breakthroughs that will enable inventions towards better future. Engineered biological systems have unique value propositions in solving the challenges through the creation of technologies that are environmentally sustainable. National Research Foundation (NRF) Published version

Accumulation of lipid-laden macrophages (foam cells) is characteristic of atherosclerosis development in the arterial walls. Ferritin nanocages have been found to passively accumulate in the atherosclerotic plaque. Ferritin has been actively investigated as a carrier for contrast agents in atherosclerosis diagnosis. We demonstrate the potential of ferritin as a carrier for therapeutic molecules to mediate cholesterol reduction from foam cells. Cyclodextrin molecules are chemically conjugated to the ferritin nanocages surface or encapsulated within the nanocages using metal co-loading methods. The cyclodextrin-conjugated ferritin has nanomolar affinity to cholesterol molecules. Treatment of foam cells with the conjugates shows decreased levels of intracellular accumulated cholesterol. The preferential localization of ferritin to foam cells is due to transferrin receptor-mediated endocytosis process. These findings show that ferritin nanocages as carriers localize cyclodextrin molecules to foam cells which mediate intracellular cholesterol reduction, thus highlighting its potential use as a therapeutic agent.

The unique properties of graphene quantum dots (GQDs) which include high loading capacity, excellent physiological stability, strong photoluminescence, biocompatibility, and facile production make them attractive nanomaterials for biomedical applications. In this work, GQDs have been explored as dual-functional targeted drug carriers and cellular bioimaging agents. The GQDs were conjugated to single chain variable fragment of antibody (scFv), which had been engineered with high affinity (B10) to epidermal growth factor receptor (EGFR), via amide covalent linkages (GQDs-scFvB10). The morphology and surface modification of GQDs were characterized by HRTEM, SDS-PAGE, FT-IR, UV-vis and fluorescence spectroscopies. Western blot analysis along with the confocal imaging of EGFR-overexpressing breast cancer cells (MDA-MB-231) demonstrated the targeting functionality of scFvB10 after conjugation to the GQDs, as well as the potential application of GQDs-scFvB10 in targeted bioimaging. The surface of targeted GQDs had a high cisplatin (CDDP) loading capacity of 50% and a pH-dependent release with slower release rate at neutral conditions, which can reduce the commonly observed systemic toxicity of CDDP. The targeted CDDP-loaded nanocarriers ((CDDP)GQDs-scFvB10) exhibited significantly higher toxicity on MDA-MB-231 cells compared to non-targeted ones suggesting their efficient uptake through EGFR. In contrast, cells with saturated EGFR showed lower uptake and cytotoxic effect of (CDDP)GQDs-scFvB10, demonstrating selectivity of the nanocarriers towards EGFR-overexpressing cells. The scFvB10-functionalized GQD is a promising platform for targeted cellular imaging and delivery of CDDP through interactions with EGFRs. This work was funded partially by NTU-Northwestern Institute for Nanomedicine at Nanyang Technological University, Singapore. The authors thank Arun Kumar Prabhakar for his assistance with the initial GQDs preparation, Barindra Sana and Valerie Loh for the reconstruction of the plasmid containing scFvB10 gene.

Circulation of cancer cells in the bloodstream is a vital step for distant metastasis, during which cancer cells are exposed to hemodynamic shear stress (SS). The actions of SS on tumor cells are complicated and not fully understood. We previously reported that fluidic SS was able to promote migration of breast cancer cells by elevating the cellular ROS level. In this study, we further investigated the mechanisms regulating SS-promoted cell migration and identified the role of MnSOD in the related pathway. We found that SS could enhance tumor cell adhesion to extracellular matrix and endothelial monolayer, and MnSOD also regulated this process. Briefly, SS stimulates the generation of mitochondrial superoxide in tumor cells. MnSOD then converts superoxide into hydrogen peroxide, which activates ERK1/2 to promote tumor cell migration and activates FAK to promote tumor cell adhesion. Combining our previous and present studies, we present experimental evidence on the pro-metastatic effects of hemodynamic SS and reveal the underlying mechanism. Our findings provide new insights into the nature of cancer metastasis and the understanding of tumor cell responses to external stresses and have valuable implications for cancer therapy development. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Accepted version

Topical approaches to oral diseases require frequent dosing due to limited wound retention times. A mucoadhesive drug delivery platform is proposed for oral wound sites with soft tissue adhesion capability of up to 7 days. Bacterial cellulose (BC) and photoactivated carbene-based bioadhesives are combined to yield flexible film platform for interfacing soft tissues in dynamic, wet environments. Structure-activity relationships evaluate UV dose and hydration state with respect to adhesive strength on soft tissue mimics. The bioadhesive composite has an adhesion strength ranging from 7-17 kPa and duration exceeding 48 hours in wet conditions under sustained shear forces, while other mucoadhesives based on hydrophilic macromolecules exhibit adhesion strength of 0.5-5 kPa and last only a few hours. The work highlights the first evaluation of BC composites for mucoadhesive treatments in the buccal cavity. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Accepted version The project is partially supported by RG17/18 (S); Wound Care Innovation for the Tropics Grant. A*STAR IAF PP Grant (H19/01/a0/ 0II9): CathoGlu Bioadhesives-preventing catheter extravasation and skin infections; Ministry of Education Tier 2 Grant: CaproGlu, Double sided wet-tissue adhesives (MOE2018-T2-2-114); Ministry of Education Tier 1 Grant: Novel light activated, diazo protecting groups.

Imaging of immune cells has wide implications in understanding disease progression and staging. While optical imaging is limited in penetration depth due to light properties, magnetic resonance (MR) imaging provides a more powerful tool for the imaging of deep tissues where immune cells reside. Due to poor MR signal to noise ratio, tracking of such cells typically requires contrast agents. This report presents an in-depth physical characterization and application of archaeal magnetoferritin for MR imaging of macrophages - an important component of the innate immune system that is the first line of defense and first responder in acute inflammation. Magnetoferritin is synthesized by loading iron in apoferritin in anaerobic condition at 65 °C. The loading method results in one order of magnitude enhancement of r

We report exceptionally large tunnel magnetoresistance (TMR) for biomolecular tunnel junctions based on ferritins immobilized between Ni and EGaIn electrodes. Ferritin stores iron in the form of ferrihydrite nanoparticles (NPs) and fulfills the following roles: (a) it dictates the tunnel barrier, (b) it magnetically decouples the NPs from the ferromagnetic (FM) electrode, (c) it stabilizes the NPs, and (d) it acts as a spin filter reducing the complexity of the tunnel junctions since only one FM electrode is required. The mechanism of charge transport is long-range tunneling which results in TMR of 60 ± 10% at 200 K and 25 ± 5% at room temperature. We propose a magnon-assisted transmission to explain the substantially larger TMR switching fields (up to 1 Tesla) than the characteristic coercive fields (a few Gauss) of ferritin ferrihydrite particles at T < 20 K. These results highlight the genuine potential of biomolecular tunnel junctions in designing functional nanoscale spintronic devices., We acknowledge the Ministry of Education (MOE) for supporting this research under award No. MOE2019-T2-1-137. Prime Minister's Office, Singapore under its Medium sized center program is also acknowledged for supporting this research. We kindly acknowledge the Singapore Synchrotron Light Source (SSLS) supporting our experiments at the SINS beam line under NUS core support C-380-003-003-001. We would like to acknowledge beamline scientists Dr Bruce Cowie and Dr Anton Tadich (soft x-ray (SXR) Beamline at the Australian Synchrotron) for their immense support. E d B acknowledges support from the US National Science Foundation (Grant No.: ECCS#1916874). S R acknowledges funding from CA2DM-NUS during his stay in Singapore. ICN2 is funded by the CERCA programme/generalitat de Catalunya, and the Severo Ochoa program from Spanish MINECO (Grant No. SEV-2017-0706).

Electrical field-induced charge modulation in graphene-based devices at the nanoscale with ultrahigh density carrier accumulation is important for various practical applications. In bilayer graphene (BLG), inversion symmetry can simply be broken by an external electric field. However, control over charge carrier density at the nanometer scale is a challenging task. Here, we demonstrate local gating of bilayer graphene (BLG) in the nanometer range by adsorption of AfFtn-AA (which is a bio-engineered ferritin, an iron storing globular protein with ∅ = 12 nm). Low-temperature electrical transport measurements with field effect transistors with these AfFtn-AA/BLG surfaces show hysteresis with two Dirac peaks. One peak at a gate voltage Vg = 35 V is associated with pristine BLG, while the second peak at Vg = 5 V results from local doping by ferritin. This charge trapping at the biomolecular length-scale offers a straightforward and non-destructive method to alter the local electronic structure of BLG.

Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits., Synthetic biological modules can be used to reprogram host proteomes, which in turn enhance the function of the synthetic modules. The authors use this holistic synthetic biology approach to engineer a more favorable environment for cell-free protein synthesis.

Summary Successful integration of proteins in solid-state electronics requires contacting them in a non-invasive fashion, with a solid conducting surface for immobilization as one such contact. The contacts can affect and even dominate the measured electronic transport. Often substrates, substrate treatments, protein immobilization, and device geometries differ between laboratories. Thus the question arises how far results from different laboratories and platforms are comparable and how to distinguish genuine protein electronic transport properties from platform-induced ones. We report a systematic comparison of electronic transport measurements between different laboratories, using all commonly used large-area schemes to contact a set of three proteins of largely different types. Altogether we study eight different combinations of molecular junction configurations, designed so that Ageoof junctions varies from 105 to 10−3 μm2. Although for the same protein, measured with similar device geometry, results compare reasonably well, there are significant differences in current densities (an intensive variable) between different device geometries. Likely, these originate in the critical contact-protein coupling (∼contact resistance), in addition to the actual number of proteins involved, because the effective junction contact area depends on the nanometric roughness of the electrodes and at times, even the proteins may increase this roughness. On the positive side, our results show that understanding what controls the coupling can make the coupling a design knob. In terms of extensive variables, such as temperature, our comparison unanimously shows the transport to be independent of temperature for all studied configurations and proteins. Our study places coupling and lack of temperature activation as key aspects to be considered in both modeling and practice of protein electronic transport experiments., Graphical Abstract, Highlights • Junction geometry determines effective contact area • Mechanism of charge transport is independent of junction platform • Electrode-molecule coupling determines transport efficiency across interfaces • Tunneling dominates solid-state electron transport across protein-based junctions, Electrochemistry; Bioelectronics; Bioelectrical Engineering

Many anti-cancer drugs are used in chemotherapy; however, little is known about their efficacy against circulating tumor cells (CTCs). In this study, we investigated whether the pulsatile fluidic shear stress (SS) in human arteries can affect the efficacy of anti-cancer drugs. Cancer cells were circulated in our microfluidic circulatory system, and their responses to drug and SS treatments were determined using various assays. Breast and cervical cancer cells that stably expressed apoptotic sensor proteins were used to determine apoptosis in real-time by fluorescence resonance energy transfer (FRET)-based imaging microscopy. The occurrence of cell death in non-sensor cells were revealed by annexin V and propidium iodide staining. Cell viability was determined by MTT assay. Intracellular reactive oxygen species (ROS) levels were determined by staining cells with two ROS-detecting dyes: 2′,7′-dichlorofluorescin diacetate and dihydroethidium. Fluidic SS significantly increased the potency of the ROS-generating drugs doxorubicin (DOX) and cisplatin but had little effect on the non-ROS-generating drugs Taxol and etoposide. Co-treatment with SS and ROS-generating drugs dramatically elevated ROS levels in CTCs, while the addition of antioxidants abolished the pro-apoptotic effects of DOX and cisplatin. More importantly, the synergistic killing effects of SS and DOX or cisplatin were confirmed in circulated lung, breast, and cervical cancer cells, some of which have a strong metastatic ability. These findings suggest that ROS-generating drugs are more potent than non-ROS-generating drugs for destroying CTCs under pulsatile fluidic conditions present in the bloodstream. This new information is highly valuable for developing novel therapies to eradicate CTCs in the circulation and prevent metastasis.

Imaging of immune cells has wide implications in understanding disease progression and staging. While optical imaging is limited in penetration depth due to light properties, magnetic resonance (MR) imaging provides a more powerful tool for the imaging of deep tissues where immune cells reside. Due to poor MR signal to noise ratio, tracking of such cells typically requires contrast agents. This report presents an in-depth physical characterization and application of archaeal magnetoferritin for MR imaging of macrophages – an important component of the innate immune system that is the first line of defense and first responder in acute inflammation. Magnetoferritin is synthesized by loading iron in apoferritin in anaerobic condition at 65 °C. The loading method results in one order of magnitude enhancement of r1 and r2 relaxivities compared to standard ferritin synthesized by aerobic loading of iron at room temperature. Detailed characterizations of the magnetoferritin revealed a crystalline core structure that is distinct from previously reported ones indicating magnetite form. The magnetite core is more stable in the presence of reducing agents and has higher peroxidase-like activities compared to the core in standard loading. Co-incubation of macrophage cells with magnetoferritin in-vitro shows significantly higher enhancement in T2-MRI contrast of the immune cells compared to standard ferritin.

Stimuli-sensitive biomaterials that are activated by light are in need of formulations that are stable under indoor lighting yet can be activated under direct sunlight. Carbene-based bioadhesives are a new generation of film-forming polymers that are stable under indoor lighting yet are rapidly activated with low-energy UVA light, but have never been evaluated under sunlight exposure. Previous investigations have evolved two flexible carbene-based platforms, where aryl-diazirine is grafted on to polyamidoamine dendrimers (PAMAM-NH2; generation-5) or hydrophobic liquid polycaprolactone tetrol to yield G5-Dzx and CaproGlu, respectively. For the first time the activation of G5-Dzx and CaproGlu is investigated by natural sunlight with intensities up to 10 mW.cm-2. Structure-property relationships of bioadhesion are investigated by: (1) joules dose of sunlight (2) bioadhesive polymer structure; and (3) optical concentrators of magnifying glass and Fresnel lens. Using only natural sunlight, adhesion strength could be tuned from 20-150 kPa with crosslinking achieved in under 1 min. The results show that carbene-based polymers are a class of stimuli-sensitive biomaterials that are stable to indoor lighting, yet can be rapidly activated under direct sunlight, which may be useful for topical film forming polymers or as active ingredients in sunscreen formulation. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Accepted version The authors would like to acknowledge the funding support that was provided by the Agency for Science, Technology and Research (A*Star, Singapore) IRG17283008 ‘Microprocessor based methods of composite curing’ & IAF PP Grant (H19/01/a0/0II9 ‘CathoGlu Bioadhesivespreventing catheter extravasation and skin infections’; Tier 1 Grant RG17/18 (S): Novel light activated, diazo protecting groups, Ministry of Education (Singapore) Tier 2 Grant (MOE2018-T2-2-114): CaproGlu, Double sided wet-tissue adhesives; and Nanyang Technological University President's Graduate Scholarship provided to Nigel C.S. Tan. This material is based upon work supported by the National Science Foundation (USA) under grant no. 1460110.

Angiogenesis marks the transformation of a benign local tumor into a life-threatening disease. Many in vitro assays are available on two-dimensional (2D) platforms, however, limited research has been conducted to investigate the behavior of tumors and endothelial cells (ECs) grown on three-dimensional (3D) platforms. This study provides a 3D co-culture spheroid of tumor cells with ECs to study the interplay between ECs and tumor cells. In a 3D co-culture with HepG2 hepatocellular carcinoma (HCC) cells, ECs differentiate to form tubule networks when in co-culture. Addition of angiogenic factors or angiogenesis inhibitors to the model system enhanced or inhibited endothelial differentiation in the 3D model, enabling investigations of the cellular signaling pathways utilized in HCC development. The 3D model demonstrated similar protein expression levels as a HCC xenograft, as well as exhibited upregulation of essential signaling proteins such as Akt/mTor in the 3D model, which is not reflected in the 2D model. The effects of several anti-angiogenic agents, such as sorafenib, sunitinib, and axitinib were analyzed in the 3D co-culture model by utilizing fluorescent proteins and a fluorescence resonance energy transfer (FRET)-based caspase-3 sensor in the ECs, which can detect apoptosis in real time. The apoptotic capability of a drug to inhibit angiogenesis in the 3D model can be easily distinguished via the FRET sensor, and dual screening of anti-angiogenesis and anti-tumor drugs can be achieved in a single step via the 3D co-culture model. In summary, a 3D co-culture model is constructed, where a HCC tumor microenvironment with a hypoxic core and true gradient penetration of drugs is achieved for drug screening purposes and in vitro studies utilizing a small HCC tumor. Biotechnol. Bioeng. 2017;114: 1865-1877. © 2017 Wiley Periodicals, Inc.

We report the development of a self-assembling protein nanocage as a contrast agent for magnetic resonance imaging (MRI). The protein nanocage is derived from genetically engineered ferritin from Archaeoglobus fulgidus (AfFtnAA). Iron (Fe) was loaded in a controlled manner within the core of the ferritin nanocage, resulting in the formation of iron oxide magnetic nanostructures (MNS). Using a variety of structural and magnetic characterization methods, we have demonstrated the magnetic and domain structures of the MNS formed within the protein nanocage and their contribution on water proton relaxation in MRI. With the primary focus on cardiac imaging for identification of atherosclerotic lesion, macrophage cell line was chosen for in vitro studies. The cytocompatibility of Fe-loaded engineered ferritin nanocages ((Fe)AfFtnAA) was confirmed by cell viability and oxidative stress measurements. The ferritin nanocages were successfully internalized by macrophage cells in a dose dependent manner and visualized under MRI. The drop in relaxation time with increasing concentration validated their potential as a contrast agent in MRI. Enhanced uptake and diagnostic capability in MRI of Fe-loaded ferritin nanocages imply their use as a “natural” probe for targeting and imaging plaque macrophages.

Ligand binding experiments between small chemicals and proteins and the evaluation of dissociation constants of their complexes in competitive binding assays often rely on displacement of reporter probes by the tested ligand. The most widely adopted protocol uses a fluorescent ligand which changes its emission spectrum when bound to a protein. A decrease of fluorescence, caused by the addition of a second ligand to the complex is generally interpreted as displacement of the fluorescent probe by the ligand, and therefore as a measure of the affinity of the ligand for the protein. Working with an odorant-binding protein (OBP), we found drastic differences in the calculated affinities when using 1-aminoanthracene or N-phenyl-1-naphthylamine as the fluorescent reporter. This fact was quite unexpected, as OBPs are small compact proteins with a single binding pocket without allosteric sites. Such observation raises doubts on the reliability of the fluorescent binding assay, perhaps the most widely used approach to evaluate affinities of small organic compounds to OBPs and other binding proteins. We recommend that the results of fluorescent binding experiments with OBPs should be confirmed by using two different probes or alternative methods. The reliability of current protocols for ligand binding assays is rather limited, while we still wait for a label-free approach that could be simple, fast and free from the use of radioactive tracers.

The mechanism of long-range charge transport across tunneling junctions with monolayers of ferritin is investigated. It is shown that the mechanism can be switched between coherent tunneling, sequential tunneling, and hopping by changing the iron content inside the ferritin. This study shows that ferritins are an interesting class of biomolecules to control charge transport.

Bacterial cellulose (BC) has been gaining importance over the past decades as a versatile material that finds applications in diverse industries. However, a secured supply is hindered by the slow production rate and batch-to-batch variability of the yield. Here, we report a rational approach for characterising the BC production process using Design of Experiment (DoE) methodology to study the impact of different parameters on desired process attributes. Notably, we found that the carbon source used for bacterial growth significantly impacts the interplay between the process variables and affects the desired outcomes. We therefore, propose that the highest priority process outcome in this study, the yield, is a function of the carbon source and optimal reactor design. Our systematic approach has achieved projected BC yields as high as ∼40 g/L for Gluconacetobacter hansenii 53582 grown on sucrose as the carbon source compared to the widely reported yields of ∼10 g/L. National Research Foundation (NRF) This research was funded by the Singapore National Research Foundation (NRF) Biological Design, Tools, and Applications (BDTA) Grant (NRF2013-THE001-046).

Bacterial cellulose (BC) is a biocompatible material with versatile applications. However, its large-scale production is challenged by the limited biological knowledge of the bacteria. The advent of synthetic biology has lead the way to the development of BC producing microbes as a novel chassis. Hence, investigation on optimal growth conditions for BC production and understanding of the fundamental biological processes are imperative. In this study, we report a novel analytical platform that can be used for studying the biology and optimizing growth conditions of cellulose producing bacteria. The platform is based on surface growth pattern of the organism and allows us to confirm that cellulose fibrils produced by the bacteria play a pivotal role towards their chemotaxis. The platform efficiently determines the impacts of different growth conditions on cellulose production and is translatable to static culture conditions. The analytical platform provides a means for fundamental biological studies of bacteria chemotaxis as well as systematic approach towards rational design and development of scalable bioprocessing strategies for industrial production of bacterial cellulose. NRF (Natl Research Foundation, S’pore) Published version

Raman spectroscopy (RS) for selective quantification of protein species in mixed solutions holds enormous potential for advancing protein detection technology to significantly faster, cheaper, and less technically demanding platforms. However, even with powerful computational methods such as nonlinear least squares regression, protein quantification in such complex systems suffers from relatively poor accuracy, especially in comparison with established methods. In this work, a combination of the expanded set of spectral information provided by polarized Raman spectroscopy (PRS) that is otherwise unavailable in conventional RS was, to our knowledge, explored to enhance the quantitative accuracy and robustness of protein quantification for the first time. A mixture containing two proteins, lysozyme and α-amylase, was used as a model system to demonstrate enhanced quantitative accuracy and robustness of selective protein quantification using PRS. The concentrations of lysozyme and α-amylase in mixtures were estimated using data obtained from both traditional RS and PRS. A new method was developed to select highly sensitive peaks for accurate concentration estimation to take advantage of additional spectra offered by PRS. The root-mean squared errors (RMSE) of estimation using traditional RS and PRS were compared. A drastic improvement in RMSE was observed from traditional RS to PRS, where the RMSEs of α-amylase and lysozyme concentrations decreased by 11 and 7 times, respectively. Therefore, this technique is a successful demonstration in achieving greater accuracy and reproducibility in the estimation of protein concentration in a mixture, and it could play a significant role in future multiplexed protein quantification platforms. Copyright © 2015 John Wiley & Sons, Ltd.

Charge separation in photosynthetic light reactions has gained much interest in an attempt to fabricate biological photovoltaic devices through integration of photosynthetic material and conducting electrodes. Direct interaction between thylakoids, as representatives of photosynthetic materials, and multi-walled carbon nanotubes (MWCNTs) is expected to increase charge transfer. Thylakoids are isolated from spinach leaf chloroplasts and pristine MWCNTs are dispersed in Triton X-100 (TX-100) as a surfactant to retain their electronic properties through non-covalent interactions. The Raman and UV-Vis spectra suggest close interactions between the thylakoids and the MWCNTs. Stable thylakoids including the embedded protein subunits and light harvesting antennas can be detected from the non-shifted 680 nm absorbance peak. The 50% fluorescence quenching in the MWCNTs-thylakoids preparation as compared to thylakoids alone using single wavelength excitation suggests charge transfer between the thylakoids and the MWCNTs.

New strategies to control cholera are urgently needed. This study develops an in vitro proof-of-concept sense-and-kill system in a wild-type Escherichia coli strain to target the causative pathogen Vibrio cholerae using a synthetic biology approach. Our engineered E. coli specifically detects V. cholerae via its quorum-sensing molecule CAI-1 and responds by expressing the lysis protein YebF-Art-085, thereby self-lysing to release the killing protein Art-085 to kill V. cholerae. For this report, we individually characterized YebF-Art-085 and Art-085 expression and their activities when coupled to our previously developed V. cholerae biosensing circuit. We show that, in the presence of V. cholerae supernatant, the final integrated sense-and-kill system in our engineered E. coli can effectively inhibit the growth of V. cholerae cells. This work represents the first step toward a novel probiotic treatment modality that could potentially prevent and treat cholera in the future.

Encapsulation of active compounds in Pickering emulsions using bioderived protein-based stabilizers holds potential for the development of novel formulations in the fields of foods and cosmetics. We employ a dodecahedron hollow protein nanocage as a pH-switchable Pickering emulsifier. E2 protein nanocages are derived from pyruvate dehydrogenase multienzyme complex from Geobacillus stearothermophilus which adsorb at the oil/water interface at neutral and basic pH's and stabilize the Pickering emulsions, while in the acidic range, at pH ∼4, the emulsion separates into emulsion and serum phases due to flocculation. The observed process is reversible for at least five cycles. Optimal formulation of a Pickering emulsion composed of rosemary oil, an essential oil, and water has been achieved by ultrasonication and results in droplets of approximately 300 nm in diameter with an oil/water ratio of 0.11 (v/v) and 0.30-0.35% (wt %). Ionic stabilization is observed for concentrations up to 250 mM NaCl and pH values from 7 to 11. The emulsions are stable for at least 10 days when stored at different temperatures up to 50 °C. The resulting Pickering emulsions of different compositions also form a gel-like structure and show shear thinning behavior under shear stress at a higher oil/water ratio.

Purpose: A ferritin-containing nanoparticle conjugated with a target-specific antibody was investigated as a MRI contrast agent for tumor detection. A genetically modified ferritin to markedly improve Fe (III) payload (up to 7,000 Fe ions), was chemically tethered to a monoclonal antibody against rat Nectin-like molecule 5 (Necl-5). Necl-5 is a cell surface glycoprotein that is highly expressed on the cell surface of many common epithelial cancers, including prostate cancer. It was previously demonstrated that this novel nanoconjugate agent exhibited effective in vitro targeting of Necl- 5 expressing tumor cells and exhibited strong MRI contrast characteristics via shortening of T 2 . Here, we demonstrate that the nanoconjugate-Necl-5 interaction can be exploited to target and detect tumor in vivo by MRI. Procedure: Using an in vivo tumor model (i.e., tumor size 0.5 -1 cm, immunodeficient beige/nude/xid mouse, xenograft injection with transformed rat prostate cells), efficacy of the conjugate targeting the tumor was examined. We used two injection strategies, a direct and a tail vein injection (0.8 mg, 300 μL per subject). Pre-injection baseline and postinjection scans were performed with the following spin-echo sequence parameters: Field of view = 90x53mm, reconstruction matrix size = 192x114, slice thickness = 1mm (10 slices), repetition time (TR) = 2070 ms, echo times (TE) = 11-198 ms in 11ms steps (18 echoes), number of averages = 2, acquisition time per scan = 7min 56s. Results: All T 2 data obtained were converted to R 2 for demonstration purposes (R 2 = 1/T 2 ). The tail vein injected conjugate significantly increased R 2 response (22.9 ± 5.2 s -1 ) as compared to control (13.5 ±1.7 s -1 ) at 4 h. The weaker R 2 increase was noted (15.2 ± 2.0 s -1 ) at 24 h. No notable changes in R 2 were observed in surrounding tissues regardless the stages of the measurement. We also measured the initial conjugate kinetics for both injection methods with respect to the ability of targeting the tumor. Direct injection of the nanoconjugate in to the center of the tumor showed a stronger and more rapid increase in R2 than the tail vein injection. Conclusion: The nanoconjugate interacts strongly and selectively in situ with Necl-5 overexpressing tumor cells. Direct injection of the nanoconjugate into the body of the tumor caused a more significant in situ R 2 increase in MRI than the tail vein injection. Varying degrees of R 2 increase within the tumor mass is likely to represent different distribution patterns of the conjugate, reflective of tumor heterogeneity.

Angiogenesis marks the transformation of a benign local tumor into a life-threatening disease. Many in vitro assays are available on two-dimensional (2D) platforms, however, limited research has been conducted to investigate the behavior of tumors and endothelial cells (ECs) grown on three-dimensional (3D) platforms. This study provides a 3D co-culture spheroid of tumor cells with ECs to study the interplay between ECs and tumor cells. In a 3D co-culture with HepG2 hepatocellular carcinoma (HCC) cells, ECs differentiate to form tubule networks when in co-culture. Addition of angiogenic factors or angiogenesis inhibitors to the model system enhanced or inhibited endothelial differentiation in the 3D model, enabling investigations of the cellular signaling pathways utilized in HCC development. The 3D model demonstrated similar protein expression levels as a HCC xenograft, as well as exhibited upregulation of essential signaling proteins such as Akt/mTor in the 3D model, which is not reflected in the 2D model. The effects of several anti-angiogenic agents, such as sorafenib, sunitinib, and axitinib were analyzed in the 3D co-culture model by utilizing fluorescent proteins and a fluorescence resonance energy transfer (FRET)-based caspase-3 sensor in the ECs, which can detect apoptosis in real time. The apoptotic capability of a drug to inhibit angiogenesis in the 3D model can be easily distinguished via the FRET sensor, and dual screening of anti-angiogenesis and anti-tumor drugs can be achieved in a single step via the 3D co-culture model. In summary, a 3D co-culture model is constructed, where a HCC tumor microenvironment with a hypoxic core and true gradient penetration of drugs is achieved for drug screening purposes and in vitro studies utilizing a small HCC tumor. Biotechnol. Bioeng. 2017;114: 1865-1877. © 2017 Wiley Periodicals, Inc.

The electrons generated by photosynthetic water splitting have been studied for direct electron transfer under light irradiation. Isolated thylakoids are incorporated as biocatalysts with indium tin oxide (ITO) as the electrode in a two-chamber photosynthetic electrochemical cell (PEC). The generated photocurrent is compared between deposited and dispersed thylakoids. The physical properties of deposited thylakoids are observed using field emission scanning electron microscopy (FESEM) and absorbance spectroscopy techniques. The results show the presence of thylakoids with bound photosystems including light harvesting antennas and other protein complexes. Further investigations reveal that the direction of electron transfer can be tuned by varying the applied potentials to promote bi-directional photocurrent. The maximum cathodic photocurrent is obtained at 50 mV vs. standard calomel electrode (SCE), while the maximum anodic photocurrent is enhanced with increasing positive potential applied. Our observation indicates the possibility of either reduction of O2 or H2O2 as the prominent cathodic photocurrent source, while electron transfer from thylakoids to ITO yields the anodic photocurrent.

Ceramic-polymer hybrid particles, intended for osteomyelitis treatment, were fabricated by preparing poly(lactic-co-glycolic acid) particles through an emulsion solvent evaporation technique, followed by calcium phosphate (CaP) coating via a surface adsorption-nucleation method. The presence of CaP coating on the surface of the particles was confirmed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. Subsequently, two antibiotics for treating bone infection, nafcillin (hydrophilic) and levofloxacin (amphiphilic), were loaded into these hybrid particles and their in vitro drug release studies were investigated. The CaP coating was shown to reduce burst release, while providing sustained release of the antibiotics for up to 4 weeks. In vitro bacterial study against Staphylococcus aureus demonstrated the capability of these antibiotic-loaded hybrid particles to inhibit biofilm formation as well as deteriorate established biofilm, making this hybrid system a potential candidate for further investigation for osteomyelitis treatment.

Bacterial cellulose (BC) is a biocompatible material with high purity and robust mechanical strength used to fabricate desirable scaffolds for 3D cell culture and wound healing. However, the chemical resistance of BC and its insolubility in the majority of solutions make it difficult to manipulate using standard chemical methods. In this study, a microfluidic process is developed to produce hollow BC microspheres with desirable internal structures and morphology. Microfluidics is used to generate a core-shell structured microparticle with an alginate core and agarose shell as a template to encapsulate Gluconacetobacter xylinus for long-term static culture. G. xylinus then secretes BC, which becomes entangled within the shell of the structured hydrogel microparticles and forms BC microspheres. The removal of the hydrogel template via thermal-chemical treatments yields robust BC microspheres exhibiting a hollow morphology. These hollow microspheres spontaneously assemble as functional units to form a novel injectable scaffold. In vitro, a highly porous scaffold is created to enable effective 3D cell culture with a high cell proliferation rate and better depth distribution. In vivo, this injectable scaffold facilitates tissue regeneration, resulting in rapid wound-healing in a Sprague Dawley rat skin model.

The exceptional chemical and physical properties of graphene oxide (GO) make it an attractive nanomaterial for biomedical applications, particularly in drug delivery. In this work we synthesized a novel, GO-based nanocarrier for the delivery of docetaxel (DTX), a potent hydrophobic chemotherapy drug. The GO was functionalized with transferrin (Tf)-poly(allylamine hydrochloride) (PAH), which provided targeted and specific accumulation to extracellular Tf receptors and stabilized GO in physiological solutions. Tf was conjugated to PAH via amide covalent linkages, and Tf-PAH coated the surface of DTX-loaded GO through electrostatic interactions. The morphology and structure of the resulting nanostructure, along with its surface modifications, were verified by use of Fourier transform infrared (FT-IR) and UV-vis spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM). DTX was loaded at a relatively high loading capacity of 37% and released in a pH-dependent and sustained manner under physiological conditions. The targeting efficiency and cytotoxicity of this drug delivery system were evaluated on MCF-7 breast cancer cells. Improved efficacy of targeted DTX-loaded nanocarrier was observed compared to nontargeted carrier and free DTX, especially at high drug concentrations. The Tf-PAH-functionalized GO nanocarrier is a promising candidate for targeted delivery and controlled release of DTX.

E2 protein cages, which consist of the assembled 60 subunits, were simulated at two levels of protonation to mimic their electrostatic properties at pH 4 and 7 using all-atom models. Starting with the initial configuration of the assembled 60-mer, either the truncation of C-terminus or the protonation state of pH 4 induces the disassembly of 60-mer, leading to the formation of trimers. Hydrodynamic radii (R h) of E2 monomer, trimer, and 60-mer are calculated from diffusivities, which agree well with experimental values. R h become smaller for the disassembled E2, confirming the formation of clusters smaller than 60-mer. Anionic Asp419 and Glu421 at the C-terminus interact with Lys240 of a neighboring trimer, while hydrophobic Leu424 and Met425 at the C-terminus interact with Pro313∼Ala318 of a neighboring trimer. When anionic residues of E2 60-mer are protonated (neutralized), the assembled 60-mer are stabilized by hydrophobic interactions of C-terminus. These indicate that the stability of E2 60-mer is attributed to both electrostatic and hydrophobic inter-trimer interactions of the neighboring C-termini. These findings support the experimental interpretation regarding the formation of trimer as an intermediate between E2 monomer and 60-mer, and help explain the key role of C-terminus for electrostatic and hydrophobic inter-trimer interactions. MOE (Min. of Education, S’pore) Accepted version

In the aromatic amino acid biosynthesis pathway, chorismate presents a branch point intermediate that is converted to tryptophan, phenylalanine (Phe), and tyrosine (Tyr). In bacteria, three enzymes catalyze the conversion of chorismate to hydroxyphenylpyruvate or pyruvate. The enzymes, chorismate mutase (CM), prephenate dehydratase (PDT), and prephenate dehydrogenase (PDHG) are either present as distinct proteins or fusions combining two activities. Gene locus AF0227 of Archaeoglobus fulgidus is predicted to encode a fusion protein, AroQ, containing all three enzymatic domains. This work describes the first characterization of a trifunctional AroQ. The A. fulgidus aroQ gene was cloned and overexpressed in Escherichia coli. The recombinant protein purified as a homohexamer with specific activities of 10, 0.51, and 50 U/mg for CM, PDT, and PDHG, respectively. Tyr at 0.5 mM concentration inhibited PDHG activity by 50%, while at 1 mM PDT was activated by 70%. Phe at 5 μM inhibited PDT activity by 66% without affecting the activity of PDHG. A fusion of CM, PDT, and PDHG domains is evident in the genome of only one other organism sequenced to date, that of the hyperthermophilic archaeon, Nanoarchaeum equitans. Such fusions of contiguous activities in a biosynthetic pathway may constitute a primitive strategy for the efficient processing of labile metabolites.