Assembly Of Biomolecules Research Articles

CONTRIBUIÇÕES DE UMA SEQUÊNCIA DIDÁTICA COM MODELOS TÁTEIS PARA AS REPRESENTAÇÕES MENTAIS DE ALUNOS UNIVERSITÁRIOS SOBRE PROTEÍNAS

Nesse estudo investigamos as representações mentais a respeito de proteínas, dentre treze alunos universitários de dois cursos da área de Ciências da Natureza, e a contribuição de uma sequência didática para a formação/reelaboração destas representações. Para tal, realizamos uma entrevista inicial, um curso de nove horas sobre proteínas e uma entrevista final. O curso foi ministrado por meio de atividades práticas com a utilização de modelos pré-fabricados para montagem de biomoléculas. Os dados coletados foram registros escritos e audiovisuais e modelos táteis de estruturas proteicas montados com materiais de baixo custo. Utilizamos a análise de conteúdo e a teoria dos Modelos Mentais de Jonhson-Laird para categorizar os alunos em modelizadores e não modelizadores e respectivas subcategorias. A sequência didática promoveu um aumento do número de alunos modelizadores, cujos modelos táteis montados e as explicações a eles associadas foram mais complexos, com detalhamento das interações intra e intermoleculares.

A Versatile Approach for the Assembly of Highly Tunable Biocatalytic Thin Films

AbstractThe deposition of protein thin films on (in)organic surfaces is a key approach to incorporate new functionalities into these materials for a broad number of applications. However, most of the current methods used for the controlled assembly of such biomolecules and eventual film formation are limiting since entail either the chemical modification of the proteins, which leads sometimes to impaired materials, or the sequential layer‐by‐layer deposition of charged macromolecules. In this work, a facile bioinspired method for the versatile fabrication of robust catalytic films is developed. The herein shown approach involves the one‐pot synthesis of porous enzyme nanogels decorated with imidazole motifs. These nanogels act as small nanoreactors that are further self‐assembled into continuous catalytic nanocoatings through metal‐imidazole coordination. An extensive structural, chemical, and functional characterization of the biocatalytic coatings evidences the integrity, stability in organic solvents and high temperatures, and the reusability of the deposits. Moreover, the thickness and metal composition of the nanocoatings can be tuned on demand. Finally, interesting applications for the bioinspired films are demonstrated, i.e., the fabrication of a glucose electrochemical biosensor and the completion of tandem enzymatic reactions, which suggest a broad applicability potential of this methodology.

Supramolecular Assembly of a Molecularly Engineered Protein and Polymer.

Programmable assembly of biomolecules is a fast growing research area that aims to emulate nature's elegance in creating numerous hierarchical self-assembled structures, which are responsible for unimaginably difficult biological functions. Protein assembly is a particularly challenging task, owing to their structural diversity, conformational heterogeneity, and high molecular weight. This article reveals the ability of a supramolecular structure-directing unit (SSDU) to regulate the entropically favourable supramolecular assembly of a covalently conjugated protein (bovine serum albumin (BSA)) to produce well-defined protein-decorated micelles with remarkably high thermal stability, suppression of the thermal denaturation of the protein, and retention of enzymatic activity. Furthermore, a SSDU-appended thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) co-assembles with the SSDU-BSA conjugate because, in both cases, assembly was primarily driven by specific molecular recognition between the SSDUs. However, the resulting supramolecular protein-polymer conjugate exhibits distinctly different polymersome structure to that of the micellar particle produced by the protein-SSDU conjugate. In this case, the enzymatic activity can be significantly suppressed above the lower critical solution temperature of supramolecularly conjugated PNIPAM, possibly due to collapse of the de-solvated polymer chains on the protein surface.

Brain Region Specific Single-Molecule Fluorescence Imaging.

We developed an approach utilizing nanoscalevesicles extracted from brain regions combined with single molecule imaging to monitor how an animal's physiological condition regulates the dynamics of protein distributions in different brain regions. This method was used to determine the effect of nicotine on the distribution of receptor stoichiometry in different mouse brain regions. Nicotine-induced upregulation of α4β2 nicotinic acetylcholine receptors (nAChRs) is associated with changes in their expression, trafficking, and stoichiometry. The structural assembly of nAChRs has been quantified in cell culture based systems using single molecule techniques. However, these methods are not capable of quantifying biomolecule assembly that takes place in a live animal. Both nicotine-induced upregulation and changes in nAChR stoichiometry differ across brain regions. Our single molecule approach revealed that nicotine acts differentially across brain regions to alter assembly in response to exposure and withdrawal.

Small-Angle Scattering as a Structural Probe for Nucleic Acid Nanoparticles (NANPs) in a Dynamic Solution Environment.

Nucleic acid-based technologies are an emerging research focus area for pharmacological and biological studies because they are biocompatible and can be designed to produce a variety of scaffolds at the nanometer scale. The use of nucleic acids (ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA)) as building materials in programming the assemblies and their further functionalization has recently established a new exciting field of RNA and DNA nanotechnology, which have both already produced a variety of different functional nanostructures and nanodevices. It is evident that the resultant architectures require detailed structural and functional characterization and that a variety of technical approaches must be employed to promote the development of the emerging fields. Small-angle X-ray and neutron scattering (SAS) are structural characterization techniques that are well placed to determine the conformation of nucleic acid nanoparticles (NANPs) under varying solution conditions, thus allowing for the optimization of their design. SAS experiments provide information on the overall shapes and particle dimensions of macromolecules and are ideal for following conformational changes of the molecular ensemble as it behaves in solution. In addition, the inherent differences in the neutron scattering of nucleic acids, lipids, and proteins, as well as the different neutron scattering properties of the isotopes of hydrogen, combined with the ability to uniformly label biological macromolecules with deuterium, allow one to characterize the conformations and relative dispositions of the individual components within an assembly of biomolecules. This article will review the application of SAS methods and provide a summary of their successful utilization in the emerging field of NANP technology to date, as well as share our vision on its use in complementing a broad suite of structural characterization tools with some simulated results that have never been shared before.

Carbon Nanotube Biohybrids: From Protein Site-Specific Coupling to the Fabrication of Reconfigurable (multi)Sensing Systems and Devices

We controlled the assembly of different biomolecules, from proteins to DNA and aptamer sequences, on carbon nanotubes (CNTs), so as to couple CNTs electronic output to biomolecular function. First we will present a facile strategy for the site-specific coupling of proteins to single walled carbon nanotubes (SWCNTs): i) in solution and with single-molecule control, with a focus on light harvesting and charge transfer investigations, as well as ii) in device configurations to investigate biomolecular (protein-protein) recognition in the context of antimicrobial resistance enzyme detection. Using an orthogonal Click reaction, Green Fluorescent Protein (GFP) was engineered to contain a genetically encoded azide group and then bound selectively to SWCNT ends in 1:1 nanohybrids with different protein orientations: in close proximity or at longer distances from the GFP’s functional center. Atomic force microscopy and fluorescence analysis in solution and on surfaces at the single-protein level confirmed the importance of bioengineering optimal protein attachment sites to achieve direct protein−nanotube communication and bridging.[1] We will then discuss the extension of this rationale into bioelectornic devices for the development of real-time biosensors with engineered protein interfacing. We assembled different variants of a b-lactamase (BL) inhibitory protein (BLIP) onto SWCNT sidewalls in electronic device configurations. We recorded the current responses in real-time for the detection of different concentrations of a class BL enzymes,[2] that degrade antibiotics, in the context of investigating antimicrobial resistance. (Resistance against the most useful antibiotics, the b-lactams, including the penicillins, is arguably the most pressing issue due to the prevalence of many different BL enzymes that degrade the antibiotic; BLIP acts as the ideal detection element in terms of its specificity to BLs and ability to bind a wide variety of different BLs). Employing a similar strategy, we will report on the fabrication of reconfigurable and solution processable nanoscale biosensing devices with multisensing capability based on SWCNTs functionalized with specific, and different, aptamer sequences employed as selective recognition elements. Distinct, hence water-soluble, SWCNT-aptamer hybrids were immobilized from solution onto different pre-patterned electrodes on the same chip, via a low-cost dielectrophoresis methodology. Multiplexed detection of three different biomarkers indicative of stress and neuro-trauma conditions was successfully performed, and real-time detection was achieved in serum down to physiologically relevant concentrations.[3] Finally, we present a strategy for the controlled assembly of stimuli-responsive end-to-end SWCNT junctions in aqueous solution, employing DNA as a molecular linker. The assembly/disassembly of the nanotubes was controlled via the intrinsic responsiveness to different stimuli of sequence-specific DNA linkers forming the junctions.[4] By and large, we demonstrate novel approaches for the designed assembly of solution-processable bioelectronic systems where single biomolecules modulate the properties of carbon nanotubes.This can yield new insights into biomolecular events down to single-molecule resolution and facilitate biomolecules’ use as integrated nanocomponents in molecular electronics and biosensing devices. [1] Journal of the American Chemical Society, 2017, 139, 17834-17840 [2] Submitted [3] Nano Letters, 2018, 18, 4130-4135 [4] Under 2nd review in Chemistry of Materials

Contrasting Chemistry of Block Copolymer Films Controls the Dynamics of Protein Self-Assembly at the Nanoscale.

Biological systems are able to control the assembly and positioning of proteins with nanoscale precision, as exemplified by the intricate molecular structures within cell membranes, virus capsids, and collagen matrices. Controlling the assembly of biomolecules is critical for the use of biomaterials in artificial systems such as antibacterial coatings, engineered tissue samples, and implanted medical devices. Furthermore, understanding the dynamics of protein assembly on heterogeneous templates will ultimately enable the control of protein crystallization in general. Here, we show a biomimetic, hierarchical bottom-up approach to direct the self-assembly of crystalline S-layers through nonspecific interactions with nanostructured block copolymer (BCP) thin-film templates. A comparison between physically and chemically patterned BCP substrates shows that chemical heterogeneity is required to confine the adhesion and self-assembly of S-layers to specific BCP domains. Furthermore, we show that this mechanism can be extended to direct the formation of collagen fibers along the principal direction of the underlying BCP substrate. The dynamics of protein self-assembly at the solid-liquid interface are followed using in situ high-resolution atomic force microscopy under continuous flow conditions, allowing the determination of the rate constants of the self-assembly. A pattern of alternating, chemically distinct nanoscale domains drastically increases the rate of self-assembly compared to non-patterned chemically homogeneous substrates.

Electrodeposition of thin chitosan membrane in freestanding SU-8 microfluidic channel for molecular addressing by capillary effect

The electrodeposition of thin chitosan membrane in freestanding microfluidic channel with capillary effect for biomolecule assembly of biosensor is investigated. A dry SU-8 sheet is used to fabricate freestanding fluidic microchannel by microelectromechanical systems (MEMS) technology. The hydrophobic surface of SU-8 microchannel transformed to hydrophilic surface by plasma treatment. The thin chitosan membrane is deposited on hydrophilic SU-8 microchannel by capillary effect, and miniaturization of sensor device is obtained. Membrane thickness of chitosan in freestanding channel can be controlled by concentration of chitosan solution and deposition time. The results show that thin chitosan membrane can be deposited by capillary effect in freestanding channel, and the deposited chitosan thin film shows great potential for immobilizing enzyme on the surface for compact biosensor device.

Supercharging enables organized assembly of synthetic biomolecules

Symmetrical protein oligomers are ubiquitous in biological systems and perform key structural and regulatory functions. However, there are few methods for constructing such oligomers. Here we have engineered completely synthetic, symmetrical oligomers by combining pairs of oppositely supercharged variants of a normally monomeric model protein through a strategy we term 'supercharged protein assembly' (SuPrA). We show that supercharged variants of green fluorescent protein can assemble into a variety of architectures including a well-defined symmetrical 16-mer structure that we solved using cryo-electron microscopy at 3.47 Å resolution. The 16-mer is composed of two stacked rings of octamers, in which the octamers contain supercharged proteins of alternating charges, and interactions within and between the rings are mediated by a variety of specific electrostatic contacts. The ready assembly of this structure suggests that combining oppositely supercharged pairs of protein variants may provide broad opportunities for generating novel architectures via otherwise unprogrammed interactions.

Chaperone-like chiral cages for catalyzing enantio-selective supramolecular polymerization

Cage catalysis has emerged as an important approach for mimicking enzymatic reactions by increasing the reaction rate and/or product selectivity of various types of covalent reactions. Here, we extend the catalytic application of cage compounds to the field of non-covalent molecular assembly. Acid-stable chiral imine cages are found to catalyze the supramolecular polymerization of porphyrins with an accelerated assembling rate and increased product enantioselectivity. Because the imine cages have a stronger interaction with porphyrin monomers and a weaker interaction with porphyrin assemblies, they can fully automatically detach from the assembled products without being consumed during the catalytic process. We reveal the kinetics of the auto-detachment of cages and the chirality growth of the assemblies using spectroscopic characterization studies. We find that the passivation groups attached to the cages are important for maintaining the structural stability of the cages during catalyzed assembly, and that the steric geometries of the cages can profoundly affect the efficiency of chiral regulation. This strategy demonstrates a new type of catalytic application of cage compounds in the field of molecular assembly, and paves the way to controlling supramolecular polymerization through a catalytic pathway.

Enzyme immobilization in completely packaged freestanding SU-8 microfluidic channel by electro click chemistry for compact thermal biosensor

Abstract Enzyme immobilization method in packaged freestanding microfluidic channel is developed for biomolecule assembly of biosensor in this research. A new micro fluidic fabrication method is proposed to fabricate freestanding fluidic structure by MEMS technology. The SU-8 polymer channel is treated by plasma treatment to change hydrophobic surface to hydrophilic property which can automatically drive the liquid into the channel by capillary force. The electro click chemistry method is carried out in hydrophilic SU-8 micro channel by capillary effect. The results show that enzyme immobilization can be immobilized by electro click chemistry in completely packaged freestanding channel. The proposed enzyme immobilization method in micro channel is capable of minimizing biosensor device for healthcare application.

Analysis of spliceosome dynamics by maximum likelihood fitting of dwell time distributions.

Colocalization single-molecule methods can provide a wealth of information concerning the ordering and dynamics of biomolecule assembly. These have been used extensively to study the pathways of spliceosome assembly in vitro. Key to these experiments is the measurement of binding times-either the dwell times of a multi-molecular interaction or times in between binding events. By analyzing hundreds of these times, many new insights into the kinetic pathways governing spliceosome assembly have been obtained. Collections of binding times are often plotted as histograms and can be fit to kinetic models using a variety of methods. Here, we describe the use of maximum likelihood methods to fit dwell time distributions without binning. In addition, we discuss several aspects of analyzing these distributions with histograms and pitfalls that can be encountered if improperly binned histograms are used. We have automated several aspects of maximum likelihood fitting of dwell time distributions in the AGATHA software package.

Mineral Surface-Templated Self-Assembling Systems: Case Studies from Nanoscience and Surface Science towards Origins of Life Research.

An increasing body of evidence relates the wide range of benefits mineral surfaces offer for the development of early living systems, including adsorption of small molecules from the aqueous phase, formation of monomeric subunits and their subsequent polymerization, and supramolecular assembly of biopolymers and other biomolecules. Each of these processes was likely a necessary stage in the emergence of life on Earth. Here, we compile evidence that templating and enhancement of prebiotically-relevant self-assembling systems by mineral surfaces offers a route to increased structural, functional, and/or chemical complexity. This increase in complexity could have been achieved by early living systems before the advent of evolvable systems and would not have required the generally energetically unfavorable formation of covalent bonds such as phosphodiester or peptide bonds. In this review we will focus on various case studies of prebiotically-relevant mineral-templated self-assembling systems, including supramolecular assemblies of peptides and nucleic acids, from nanoscience and surface science. These fields contain valuable information that is not yet fully being utilized by the origins of life and astrobiology research communities. Some of the self-assemblies that we present can promote the formation of new mineral surfaces, similar to biomineralization, which can then catalyze more essential prebiotic reactions; this could have resulted in a symbiotic feedback loop by which geology and primitive pre-living systems were closely linked to one another even before life’s origin. We hope that the ideas presented herein will seed some interesting discussions and new collaborations between nanoscience/surface science researchers and origins of life/astrobiology researchers.

The Recombination Mediator BRCA2: Architectural Plasticity of Recombination Intermediates Revealed by Single-Molecule Imaging (SFM/TIRF).

Cellular functions are defined by dynamic assembly, rearrangement, and disassembly of biomolecules to achieve control and specificity. As an example, effective DNA repair is brought about by the concerted action of several DNA processing proteins. Both changes in the structure of individual proteins and in the arrangement of multiple proteins together (referred to here as architecture) are inherent to biological function. These dynamic changes are exemplified in the breast cancer susceptibility protein 2 (BRCA2). BRCA2 is a DNA repair protein that undergoes changes in its own structure and affects changes in molecular architecture with partners during homologous recombination (HR) repair of DNA double strand breaks. These challenging features of BRCA2 protein, its size and predicted stretches of intrinsically disordered regions, have made it difficult to determine the structural consequences and mechanistic importance of interactions between full-length BRCA2 with RAD51 and other HR proteins. In this chapter, we describe scanning force microscopy (SFM)-based approaches to study DNA-protein complexes involved in HR, the architectural plasticity of full-length BRCA2, and the dynamic reorganization of these molecular components associated with essential steps of HR.

A dual-amplification system for colorimetric DNA detection based on the assembly of biomolecules

A dual-amplification system is reported to apply in DNA sensing via the assembly of DNA and protein. In this process, the biotinylatedcapature DNA bounded with streptavidin (SA) through the biotin-streptavidin reaction, and then the assembly of DNA and protein was triggered by the linker DNA after the target hybridized with biotinylatedcapature DNA. Sequentially, the 3,3',5,5'-tetramethylbenzidine (TMB) was oxidized by H2O2 under the catalysis of horseradish peroxidase. Based on the variation of the color and the UV–vis absorbance intensities, qualitative and quantitative DNA analyses were realized. This proposed method could detect the target DNA as low as 1.75pmol/L and discriminate perfectly matched target DNA from the mismatch DNA. What’s more, it can be expanded to detect other molecules with a reasonable design of the corresponding DNA sequences.

"Martinizing" the Variational Implicit Solvent Method (VISM): Solvation Free Energy for Coarse-Grained Proteins.

Solvation is a fundamental driving force in many biological processes including biomolecular recognition and self-assembly, not to mention protein folding, dynamics, and function. The variational implicit solvent method (VISM) is a theoretical tool currently developed and optimized to estimate solvation free energies for systems of very complex topology, such as biomolecules. VISM’s theoretical framework makes it unique because it couples hydrophobic, van der Waals, and electrostatic interactions as a functional of the solvation interface. By minimizing this functional, VISM produces the solvation interface as an output of the theory. In this work, we push VISM to larger scale applications by combining it with coarse-grained solute Hamiltonians adapted from the MARTINI framework, a well-established mesoscale force field for modeling large-scale biomolecule assemblies. We show how MARTINI-VISM (MVISM) compares with atomistic VISM (AVISM) for a small set of proteins differing in size, shape, and charge distribution. We also demonstrate MVISM’s suitability to study the solvation properties of an interesting encounter complex, barnase–barstar. The promising results suggest that coarse-graining the protein with the MARTINI force field is indeed a valuable step to broaden VISM’s and MARTINI’s applications in the near future.

Stable optical trapping and sensitive characterization of nanostructures using standing-wave Raman tweezers

Optical manipulation and label-free characterization of nanoscale structures open up new possibilities for assembly and control of nanodevices and biomolecules. Optical tweezers integrated with Raman spectroscopy allows analyzing a single trapped particle, but is generally less effective for individual nanoparticles. The main challenge is the weak gradient force on nanoparticles that is insufficient to overcome the destabilizing effect of scattering force and Brownian motion. Here, we present standing-wave Raman tweezers for stable trapping and sensitive characterization of single isolated nanostructures with a low laser power by combining a standing-wave optical trap with confocal Raman spectroscopy. This scheme has stronger intensity gradients and balanced scattering forces, and thus can be used to analyze many nanoparticles that cannot be measured with single-beam Raman tweezers, including individual single-walled carbon nanotubes (SWCNT), graphene flakes, biological particles, SERS-active metal nanoparticles, and high-refractive semiconductor nanoparticles. This would enable sorting and characterization of specific SWCNTs and other nanoparticles based on their increased Raman fingerprints.

Molecular Tools and Nanodevices Built from DNA

Over the last decades, DNA has proven to be an excellent choice for the designed self-assembly of functional two- and three-dimensional nanostructures and materials [1,2]. By offering attachment sites for active nano-components on DNA-based objects, our group has realized complex and nanometer-precise assemblies of fluorophores, plasmonic nanoparticles and biomolecules [3].

“Parking-garage” structures in nuclear astrophysics and cellular biophysics

A striking shape was recently observed for the cellular organelle endoplasmic reticulum consisting of stacked sheets connected by helical ramps. This shape is interesting both for its biological function, to synthesize proteins using an increased surface area for ribosome factories, and its geometric properties that may be insensitive to details of the microscopic interactions. In the present work, we find very similar shapes in our molecular dynamics simulations of the nuclear pasta phases of dense nuclear matter that are expected deep in the crust of neutron stars. There are dramatic differences between nuclear pasta and terrestrial cell biology. Nuclear pasta is 14 orders of magnitude denser than the aqueous environs of the cell nucleus and involves strong interactions between protons and neutrons, while cellular scale biology is dominated by the entropy of water and complex assemblies of biomolecules. Nonetheless the very similar geometry suggests both systems may have similar coarse-grained dynamics and that the shapes are indeed determined by geometrical considerations, independent of microscopic details. Many of our simulations self-assemble into flat sheets connected by helical ramps. These ramps may impact the thermal and electrical conductivities, viscosity, shear modulus, and breaking strain of neutron star crust. The interaction we use, with Coulomb frustration, may provide a simple model system that reproduces many biologically important shapes.

On-Electrode Synthesis of Shape-Controlled Hierarchical Flower-Like Gold Nanostructures for Efficient Interfacial DNA Assembly and Sensitive Electrochemical Sensing of MicroRNA.

The performance for biomolecular detection is closely associated with the interfacial structure of a biosensor, which profoundly affects both thermodynamics and kinetics of the assembly, binding and signal transduction of biomolecules. Herein, it is reported on a one-step and template-free on-electrode synthesis method for making shape-controlled gold nanostructures on indium tin oxide substrates, which provide an electrochemical sensing platform for ultrasensitive detection of nucleic acids. Thus-prepared hierarchical flower-like gold nanostructures (HFGNs) possess large surface area that can readily accommodate the assembly of DNA probes for subsequent hybridization detection. It is found that the sensitivity for electrochemical DNA sensing is critically dependent on the morphology of HFGNs. By using this new strategy, a highly sensitive electrochemical biosensor is developed for label-free detection of microRNA-21 (miRNA-21), a biomarker for lung cancers. Importantly, it is demonstrated that this biosensor can be employed to measure the miRNA-21 expression level from human lung cancer cell (A549) lysates and worked well in 100% serum, suggesting its potential for applications in clinical diagnosis and a wide range of bioanalysis.