Continued dependence on crude oil and natural gas resources for fossil fuels has caused global atmospheric carbon dioxide (CO2) emissions to increase to record-setting proportions. There is an urgent need for efficient and inexpensive carbon sequestration systems to mitigate large-scale CO2 emissions from industrial flue gas. Carbonic anhydrase (CA) has shown high potential for enhanced CO2 capture applications compared to conventional absorption-based methods currently utilized in various industrial settings. This study aims to understand structural aspects that contribute to the stability of CA enzymes critical for their applications in industrial processes, which require the ability to withstand conditions different from their native environments. Here, we evaluated the thermostability and enzyme activity of mesophilic and thermophilic CA variants at different temperature conditions and in the presence of atmospheric gas pollutants like nitrogen oxides (NOx) and sulphur oxides (SOx). Based on our enzyme activity assays and molecular dynamics simulations, we see increased conformational stability and CA activity levels in thermostable CA variants incubated week-long at different temperature conditions. The thermostable CA variants also retained high levels of CA activity despite changes in solution pH due to increasing NOx and SOx concentrations. Furthermore, a loss of CA activity was observed only at high concentrations of NOx/SOx that possibly can be minimized with appropriate buffered solutions.
Filamentous fungi can synthesize a variety of nanoparticles (NPs), a process referred to as mycosynthesis that requires little energy input, do not require the use of harsh chemicals, occurs at near neutral pH, and do not produce toxic byproducts. While NP synthesis involves reactions between metal ions and exudates produced by the fungi, the chemical and biochemical parameters underlying this process remain poorly understood. Here, the role of fungal species and precursor salt on the mycosynthesis of zinc oxide (ZnO) NPs is investigated. This data demonstrates that all five fungal species tested are able to produce ZnO structures that can be morphologically classified into i) well-defined NPs, ii) coalesced/dissolving NPs, and iii) micron-sized square plates. Further, species-dependent preferences for these morphologies are observed, suggesting potential differences in the profile or concentration of the biochemical constituents in their individual exudates. This data also demonstrates that mycosynthesis of ZnO NPs is independent of the anion species, with nitrate, sulfate, and chloride showing no effect on NP production. Finally, these results enhance the understanding of factors controlling the mycosynthesis of ceramic NPs, supporting future studies that can enable control over the physical and chemical properties of NPs formed through this “green” synthesis method.
Intracellular transport by kinesin motors moving along their associated cytoskeletal filaments, microtubules, is essential to many biological processes. This active transport system can be reconstituted in vitro with the surface-adhered motors transporting the microtubules across a planar surface. In this geometry, the kinesin-microtubule system has been used to study active self-assembly, to power microdevices, and to perform analyte detection. Fundamental to these applications is the ability to characterize the interactions between the surface tethered motors and microtubules. Fluorescence Interference Contrast (FLIC) microscopy can illuminate the height of the microtubule above a surface, which, at sufficiently low surface densities of kinesin, also reveals the number, locations, and dynamics of the bound motors.
Fungi produce and excrete various proteins, enzymes, and polysaccharides, which may be used for the synthesis of nanoparticles. This study investigated the effect an anion species on the synthesis of ceramic nanoparticles using fungal filtrates. In this work, ceramic zinc oxide (ZnO) nanoparticles ranging between 1 nm and 1000 nm were successfully synthesized using three different filamentous fungi: Aspergillus sp., Penicillium sp., and Paecilomyces variotti. Each fungus was cultured, and the filtrate was extracted and individually exposed to zinc nitrate, zinc sulfate, or zinc chloride. The formation of nanoparticles was characterized using UV-visible spectrophotometry (UV-Vis), fluorescence microscopy, and with transmission electron microscopy (TEM) analyses. UV-Vis spectra exhibited broad increases in the absorption across the range of 200 nm - 800 nm, which corresponded to the formation of ZnO nanoparticles under various conditions. Nanoparticle formation was confirmed with fluorescence microscopy and TEM analysis and determined to form particles with an irregular spherical shape. To date, our work demonstrates that the ability of fungi to synthesize ZnO nanoparticles is not genus/species-specific but is dependent on the starting composition of a given metal salt.
Dynamic instability of microtubules is characterized by stochastically alternating phases of growth and shrinkage and is hypothesized to be controlled by the conformation and nucleotide state of tubulin dimers within the microtubule lattice. Specifically, conformation changes (compression) in the tubulin dimer following the hydrolysis of GTP have been suggested to generate stress and drive depolymerization. In the present study, molecular dynamics simulations were used in tandem with in vitro experiments to investigate changes in depolymerization based on the presence of islands of uncompressed (GMPCPP) dimers in the microtubule lattice. Both methods revealed an exponential decay in the kinetic rate of depolymerization corresponding to the relative level of uncompressed (GMPCPP) dimers, beginning at approximately 20% incorporation. This slowdown was accompanied by a distinct morphological change from unpeeling “ram’s horns” to blunt-ended dissociation at the microtubule end. Collectively these data demonstrated that islands of uncompressed dimers can alter the mechanism and kinetics of depolymerization in a manner consistent with promoting rescue events.
Boronic acid-modified polymers (BAMPs) can interact with glycoproteins and other glycosylated compounds through covalent binding of the boronic acid moieties to saccharide residues. As a first step toward evaluating the utility of BAMPs as SARS-CoV-2 antiviral agents, this COVID-19 rapid response LDRD was intended to examine the effect of BAMPs on SARS-CoV-2 spike glycoprotein and its subsequent binding with ACE2 receptor protein. Multiple different approaches were attempted in order to determine whether BAMPs based on poly(ethylene glycol) and poly(ethylenimine) bind the spike protein, but failed to produce a definitive answer. However, two different enzyme-linked immunosorbent assays clearly showed no discernable effect of boronic acid in inhibiting spike-ACE2 binding.
Nuclear pore complexes (NPCs) regulate all cargo traffic across the nuclear envelope. The transport conduit of NPCs is highly enriched in disordered phenylalanine/glycine-rich nucleoporins (FG-Nups), which form a permeability barrier of still elusive and highly debated molecular structure. Here we present a microfluidic device that triggered liquid-to-liquid phase separation of FG-Nups, which yielded droplets that showed typical properties of a liquid state. On the microfluidic chip, droplets were perfused with different transport-competent or -incompetent cargo complexes, and then the permeability barrier properties of the droplets were optically interrogated. We show that the liquid state mimics permeability barrier properties of the physiological nuclear transport pathway in intact NPCs in cells: that is, inert cargoes ranging from small proteins to large capsids were excluded from liquid FG-Nup droplets, but functional import complexes underwent facilitated import into droplets. Collectively, these data provide an experimental model of how NPCs can facilitate fast passage of cargoes across an order of magnitude in cargo size.
Cytoskeletal filaments and motor proteins are critical components in the transport and reorganization of membrane-based organelles in eukaryotic cells. Previous studies have recapitulated the microtubule-kinesin transport system in vitro to dynamically assemble large-scale nanotube networks from multilamellar liposomes and polymersomes. Moving toward more biologically relevant systems, the present work examines whether lipid nanotube (LNT) networks can be generated from giant unilamellar vesicles (GUVs) and subsequently characterizes how the lipid composition may be tuned to alter the dynamics, structure, and fluidity of networks. Here, we describe a two-step process in which microtubule motility (i) drives the transport and aggregation of GUVs to form structures with a decreased energy barrier for LNT formation and (ii) extrudes LNTs without destroying parent GUVs, allowing for the formation of large LNT networks. We further show that the lipid composition of the GUV influences formation and morphology of the extruded LNTs and associated networks. For example, LNTs formed from phase-separated GUVs (e.g., liquid-solid phase-separated and coexisting liquid-ordered and liquid-disordered phase-separated) display morphologies related to the specific phase behavior reflective of the parent GUVs. Overall, the ability to form nanotubes from compositionally complex vesicles opens the door to generating lipid networks that more closely mimic the structure and function of those found in cellular systems.
Kinesin motors and their associated filaments, microtubules, are essential to many biological processes. The motor and filament system can be reconstituted in vitro with the surface-adhered motors transporting the filaments along the surface. In this format, the system has been used to study active self-assembly and to power microdevices or perform analyte detection. However, fundamental properties of the system, such as the spacing of the kinesin motors bound to the microtubule and the dynamics of binding, remain poorly understood. We show that Fluorescence Interference Contrast (FLIC) microscopy can illuminate the exact height of the microtubule, which for a sufficiently low surface density of kinesin, reveals the locations of the bound motors. We examine the spacing of the kinesin motors on the microtubules at various kinesin surface densities and compare the results with theory. FLIC reveals that the system is highly dynamic, with kinesin binding and unbinding along the length of the microtubule as it is transported along the surface.
Structural defects can determine and influence various properties of materials, and many technologies rely on the manipulation of defects (e.g., semiconductor industries). In biological systems, management of defects/errors (e.g. DNA repair) is critical to an organism's survival, which has inspired the design of artificial nanomachines that mimic nature's ability to detect defects and repair damage. Biological motors have captured considerable attention in developing such capabilities due to their ability to convert energy into directed motion in response to environmental stimuli, which maximizes their ability for detection and repair. The objective of the present study was to develop an understanding of how the presence of non-bonding domains, here considered as a "defect", in microtubule (MT) building blocks affect the kinesin-driven, active assembly of MT spools. The assembly/joining of micron-scale bonding (i.e., biotin-containing) and non-bonding (i.e., no biotin) MTs resulted in segmented MT building blocks consisting of alternating bonding and non-bonding domains. Here, the introduction of these MT building blocks into a kinesin gliding motility assay along with streptavidin-coated quantum dots resulted in the active assembly of spools with altered morphology but retained functionality. Moreover, it was noted that non-bonding domains were autonomously and preferentially released from the spools over time, representing a mechanism by which defects may be removed from these structures. Overall, our findings demonstrate that this active assembly system has an intrinsic ability for quality control, which can be potentially expanded to a wide range of applications such as self-regulation and healing of active materials.
We show that olefin metathesis can be used in an extremely simple process to rapidly alter the morphology of self-assembled poly(butadiene-b-ethylene oxide) (PB-PEO) dispersions in situ. The addition of a water-insoluble Hoveyda-Grubbs catalyst to aqueous assemblies of PB-PEO leads to degradation of the hydrophobic PB block by well-established metathesis pathways and a concomitant change in the composition of the block copolymer. This phenomenon drives morphological transitions characterized by rapidly decreasing sizes of the self-assembled aggregates, the ultimate extent of which is readily controlled by catalyst concentration. Exemplary cases are presented in which transitions from worm-like micelles to spherical micelles or from vesicles to worm-like micelles can be accomplished within minutes.
Microtubule dynamics play a critical role in the normal physiology of eukaryotic cells as well as a number of cancers and neurodegenerative disorders. The polymerization/depolymerization of microtubules is regulated by a variety of stabilizing and destabilizing factors, including microtubule-associated proteins and therapeutic agents (e.g., paclitaxel, nocodazole). Here we describe the ability of the osmolytes polyethylene glycol (PEG) and trimethylamine-N-oxide (TMAO) to inhibit the depolymerization of individual microtubule filaments for extended periods of time (up to 30 days). We further show that PEG stabilizes microtubules against both temperature- and calcium-induced depolymerization. Our results collectively suggest that the observed inhibition may be related to combination of the kosmotropic behavior and excluded volume/osmotic pressure effects associated with PEG and TMAO. Taken together with prior studies, our data suggest that the physiochemical properties of the local environment can regulate microtubule depolymerization and may potentially play an important role in in vivo microtubule dynamics.
The directed, head-to-tail self-assembly of microtubule filaments may be generalized in the context of Janus colloidal rods. Specifically, their assembly at the tens of micron-length scale involves a careful balance between long-range electrostatic repulsion and short-range attractive forces. Here we show that the addition of counterion salts increases the rate of directed assembly by screening the electrostatic forces and enhancing the effectiveness of short-range interactions at the microtubule ends.
The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.
The selective partitioning of lipid components in phase-separated membranes is essential for domain formation involved in cellular processes. Identifying and tracking the movement of lipids in cellular systems would be improved if we understood how to achieve selective affinity between fluorophore-labeled lipids and membrane assemblies. Here, we investigated the structure and chemistry of membrane lipids to evaluate lipid designs that partition to the liquid ordered (Lo) phase. A range of fluorophores at the headgroup position and lengths of PEG spacer between the lipid backbone and fluorophore were examined. On a lipid body with saturated palmityl or palmitoyl tails, we found that although the lipid tails can direct selective partitioning to the Lo phase through favorable packing interactions, headgroup hydrophobicity can override the partitioning behavior and direct the lipid to the disordered membrane phase (Ld). The PEG spacer can serve as a buffer to mute headgroup-membrane interactions and thus improve Lo phase partitioning, but its effect is limited with strongly hydrophobic fluorophore headgroups. We present a series of lipid designs leading to the development of novel fluorescently labeled lipids with selective affinity for the Lo phase.
Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.). Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol)-poly(butadiene) (PEO-PBD) polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased by increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm). This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Overall the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.
Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (> 4 μm) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (>4 μm) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (>100 μm).
Nanoscale control of matter is critical to the design of integrated nanosystems. Here, we describe a method to dynamically control directionality of microtubule (MT) motion using programmable magnetic fields. MTs are combined with magnetic quantum dots (i.e., MagDots) that are manipulated by external magnetic fields provided by magnetic nanowires. MT shuttles thus undergo both ATP-driven and externally-directed motion with a fluorescence component that permits simultaneous visualization of shuttle motion. This technology is used to alter the trajectory of MTs in motion and to pin MT motion. Such an approach could be used to evaluate the MT-kinesin transport system and could serve as the basis for improved lab-on-a-chip technologies based on MT transport.
Active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly. Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications.
Molecular motor-driven self-assembly has been an active area of soft matter research for the past decade. Because molecular motors transform chemical energy into mechanical work, systems which employ molecular motors to drive self-assembly processes are able to overcome kinetic and thermodynamic limits on assembly time, size, complexity, and structure. Here, we review the progress in elucidating and demonstrating the rules and capabilities of motor-driven active self-assembly. We focus on the types of structures created and the degree of control realized over these structures, and discuss the next steps necessary to achieve the full potential of this assembly mode which complements robotic manipulation and passive self-assembly.
This report describes a new, bio-inspired approach to electrical energy storage, based on the generation, maintenance, and discharge of ion concentration gradients. This approach was investigated as part of a Laboratory Directed Research and Development program at Sandia National Laboratories in Albuquerque, NM between October 2012 and September 2015. In particular, this report describes the development of ion-selective nanoporous membranes, controlled through pore morphology and Sandia-developed electrochemical functionalization. In addition, it describes a potential avenue to functionalize synthetic polymer membranes in a way that facilitates oriented ion pump binding to polymer surfaces. Finally, it highlights a number of new computational findings central to the understanding and ultimate design of synthetic ion channels and ion gates needed for biomimetic ion-based electrochemical energy storage.
Despite significant progress in development of bioanalytical devices cost, complexity, access to reagents and lack of infrastructure have prevented use of these technologies in resource-limited regions. To provide a sustainable tool in the global effort to combat infectious diseases the diagnostic device must be low cost, simple to operate and read, robust, and have sensitivity and specificity comparable to laboratory analysis. In this mini-review we describe recent work using laser machined plastic laminates to produce diagnostic devices that are capable of a wide variety of bioanalytical measurements and show great promise towards future use in low-resource environments.
In this study, we describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4–5 h for corresponding lipid networks). The transport of materials in and on the polymer membranes differs substantially from the transport on analogous lipid networks. Specifically, our data suggest that polymer mobility in nanotubular structures is considerably different from planar or 3D structures, and is stunted by 1D confinement of the polymer subunits. Moreover, quantum dots adsorbed onto polymer nanotubes are completely immobile, which is related to this 1D confinement effect and is in stark contrast to the highly fluid transport observed on lipid tubules.
For more than a decade now, biomolecular systems have served as an inspiration for the development of synthetic nanomaterials and systems that are capable of reproducing many of unique and emergent behaviors of living systems. In addition, one intriguing element of such systems may be found in a specialized class of proteins known as biomolecular motors that are capable of performing useful work across multiple length scales through the efficient conversion of chemical energy. Microtubule (MT) filaments may be considered within this context as their dynamic assembly and disassembly dissipate energy, and perform work within the cell. MTs are one of three cytoskeletal filaments in eukaryotic cells, and play critical roles in a range of cellular processes including mitosis and vesicular trafficking. Based on their function, physical attributes, and unique dynamics, MTs also serve as a powerful archetype of a supramolecular filament that underlies and drives multiscale emergent behaviors. In this review, we briefly summarize recent efforts to generate hybrid and composite nanomaterials using MTs as biomolecular scaffolds, as well as computational and synthetic approaches to develop synthetic one-dimensional nanostructures that display the enviable attributes of the natural filaments.
Polycarbonate is a desirable material for many applications due to its favorable mechanical and optical properties. Here, we report a simple, safe, environmentally friendly aqueous method that uses diamines to functionalize a polycarbonate surface with amino groups. The use of water as the solvent for the functionalization ensures that solvent induced swelling does not affect the optical or mechanical properties of the polycarbonate. We characterize the efficacy of the surface amination using X-ray photo spectroscopy, Fourier transform infrared spectroscopy (FT-IR), atomic force microscopy (AFM), and contact angle measurements. Furthermore, we demonstrate the ability of this facile method to serve as a foundation upon which other functionalities may be attached, including antifouling coatings and oriented membrane proteins. (Chemical Presented).
Anthrax poses a significant threat to National Security as demonstrated by the terrorist attacks targeting the US Postal Service and Hart Building. Anthrax outbreaks commonly occur in livestock. Consequently, Bacillus anthracis is routinely isolated, propagated, and maintained to diagnose the disease. This practice increases laboratories' repositories of the agent, escalating the risk that it can be stolen. We have developed BaDX (2014 R&D100 Awardee), a credit-card sized diagnostic device for use in ultra-low resource environments that is low cost, requires no power, instrumentation or equipment to operate, no cold chain, self-decontaminates post-assay, and is operable by individuals with little/no technical training.