Macromolecules Of Interest Research Articles

Selective signal enhancement in Fourier space as a tool for discovering ultrastructural organization of macromolecules from in situ TEM

We present a Fourier transform (FT) based analytical method that allows to obtain of ultrastructural details from TEM images at sub-nanometer scale applying a selective filtering for singular macromolecule electron microscopy density information. It can be applied to high-pressure frozen, frozen hydrated and epoxy freeze substituted and embedded biological species. Both 2D projections and orthoslices from reconstructed tomograms can be used as a source of structural information. The key to the method is to select the macromolecule or organelle of interest with an accuracy of ≥ 7 – 3 nm (depending on pixel size of initial tilt series or singular image acquisition) and explore both the central low frequency FT intensity and diffraction regions to obtain the spatial structural organization and its dimensional characteristics, respectively. We also introduce a structure-specific selective mask FT filtering approach that can significantly improve image information even in poorly contrasted TEM of resin sections without heavy metal been used. The described method elucidates chromatin architecture without the need of averaging. A zigzag symmetry of 30 nm diameter chromatin fibers which in general is a controversial topic of research has been identified for C. elegans cells in vivo with sub-nanometer details being preserved in the images.

Engineering highly stable variants of Corynactis californica green fluorescent proteins.

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

Structural Characterization of Biomass from Crassiphycus birdiae in natura and Residual

Macroalgae contains micro and macromolecules of great interest for various sectors, for example, food, cosmetics, and pharmaceuticals. These compounds can be obtained through different extraction methods, among them the use of organic solvents with varying polarities. In this study, materials were characterized using scanning electron microscopy and energy-dispersive X-ray spectroscopy, infrared spectrometry, X-ray diffraction and differential scanning calorimetry in the seaweed biomasses before and after an extraction process with organic solvents. The red macroalgae Crassiphycus birdiae, cultivated on the northeast coast of Brazil, appeared, after extraction, as an amorphous material with some porosity. Its composition includes the elements carbon, oxygen, sodium, magnesium, sulfur, chlorine, potassium, aluminum, silicon, calcium, and iron. The presence of the agar polysaccharide was also verified by infrared spectroscopy. The decomposition of this polysaccharide was observed using differential scanning calorimetry.

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

Crowder titrations enable the quantification of driving forces for macromolecular phase separation

Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. In addition, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation.

Xylan Solubilization from Partially Delignified Biomass, and Residual Lignin Removal from Solubilized Xylan

Xylan is a macromolecule of industrial interest that can be solubilized from lignocellulosic materials, such as sugarcane bagasse, which is a renewable source. However, the solubilization methods of xylan need to be better developed for use in industrial applications. The main objective of this study was to evaluate xylan solubilization methods with higher yields and purity by using biomasses/fractions of sugarcane: leaf and stem, internode, node, and external fraction. Two strategies were evaluated by applying diluted sodium chlorite, sodium sulfite, and hydrogen peroxide: a delignification of the biomass before xylan solubilization; and the delignification of the solubilized xylan for residual lignin removal. The delignification of the biomass before the xylan solubilization enabled to identify material and specific conditions for yields higher than 90%. Residual lignin varied from 3.14 to 18.06%, with hydrogen peroxide in alkaline medium partial delignification shown to be effective. The delignification of xylan presented better results using diluted hydrogen peroxide, with a reduction of 58.44% of the initial lignin content. The solubilized xylans were used as a substrate for xylanase activities, resulting in higher activity than commercial xylan. In the delignification of the biomasses, hydrogen peroxide was the reagent with better results concerning the yield, purity, and solubility of the xylan. This reagent (diluted) was also better in the delignification of the solubilized xylan, resulting in lower residual lignin content. The solubility and purity tests (low salt content) indicated that the solubilized xylan presented characteristics that were similar to or even better than commercial xylan.

Spotlight on the Winners of the DCO‐SCF 2021 Awards – An Ever Valuable Collaboration with EurJOC

Spotlight on the Winners of the DCO‐SCF 2021 Awards – An Ever Valuable Collaboration with EurJOC

Quantifying Coexistence Concentrations in Multi-Component Phase-Separating Systems Using Analytical HPLC.

Over the last decade, evidence has accumulated to suggest that numerous instances of cellular compartmentalization can be explained by the phenomenon of phase separation. This is a process by which a macromolecular solution separates spontaneously into dense and dilute coexisting phases. Semi-quantitative, in vitro approaches for measuring phase boundaries have proven very useful in determining some key features of biomolecular condensates, but these methods often lack the precision necessary for generating quantitative models. Therefore, there is a clear need for techniques that allow quantitation of coexisting dilute and dense phase concentrations of phase-separating biomolecules, especially in systems with more than one type of macromolecule. Here, we report the design and deployment of analytical High-Performance Liquid Chromatography (HPLC) for in vitro separation and quantification of distinct biomolecules that allows us to measure dilute and dense phase concentrations needed to reconstruct coexistence curves in multicomponent mixtures. This approach is label-free, detects lower amounts of material than is accessible with classic UV-spectrophotometers, is applicable to a broad range of macromolecules of interest, is a semi-high-throughput technique, and if needed, the macromolecules can be recovered for further use. The approach promises to provide quantitative insights into the balance of homotypic and heterotypic interactions in multicomponent phase-separating systems.

On the Interfacial Behavior of Catenated Poly(l-lactide) at the Air–Water Interface

Interfacial properties of polymeric materials are significantly influenced by their architectural structures and spatial features, while such a study of topologically interesting macromolecules is rarely reported. In this work, we reported, for the first time, the interfacial behavior of catenated poly(l-lactide) (C-PLA) at the air-water interface and compared it with its linear analogue (L-PLA). The isotherms of surface pressure-area per repeating unit showed significant interfacial behavioral differences between the two polymers with different topologies. Isobaric creep experiments and compression-expansion cycles also showed that C-PLA demonstrated higher stability at the air-water interface. Interestingly, when the films at different surface pressures were transferred via the Langmuir-Blodgett method, successive atomic force microscopy imaging displayed distinct nanomorphologies, in which the surface of C-PLA exhibited nanofibrous structures, while that of the L-PLA revealed a smoother topology with less fiber-like structures.

Self-consistent field theory of chiral nematic worm-like chains.

Many macromolecules of biological and technological interest are both chiral and semi-flexible. DNA and collagen are good examples. Such molecules often form chiral nematic (or cholesteric) phases, as is well-documented in collagen and chitin. This work presents a method for studying cholesteric phases in the highly successful self-consistent field theory of worm-like chains, offering a new way of studying many biologically relevant molecules. The method involves an effective Hamiltonian with a chiral term inspired by the Oseen-Frank (OF) model of liquid crystals. This method is then used to examine the formation of cholesteric phases in chiral-nematic worm-like chains as a function of polymer flexibility, as well as the optimal cholesteric pitch and distribution of polymer segment orientations. Our approach not only allows for the determination of the isotropic-cholesteric transition and segment distributions, beyond what the OF model promises, but also explicitly incorporates polymer flexibility into the study of the cholesteric phase, offering a more complete understanding of the behavior of semiflexible chiral-nematic polymers.

A setup for millisecond time-resolved X-ray solution scattering experiments at the CoSAXS beamline at the MAX IV Laboratory.

The function of biomolecules is tightly linked to their structure, and changes therein. Time-resolved X-ray solution scattering has proven a powerful technique for interrogating structural changes and signal transduction in photoreceptor proteins. However, these only represent a small fraction of the biological macromolecules of interest. More recently, laser-induced temperature jumps have been introduced as a more general means of initiating structural changes in biomolecules. Here we present the development of a setup for millisecond time-resolved X-ray solution scattering experiments at the CoSAXS beamline, primarily using infrared laser light to trigger a temperature increase, and structural changes. We present results that highlight the characteristics of this setup along with data showing structural changes in lysozyme caused by a temperature jump. Further developments and applications of the setup are alsodiscussed.

Cryo-EM Analyses Permit Visualization of Structural Polymorphism of Biological Macromolecules.

The functions of biological macromolecules are often associated with conformational malleability of the structures. This phenomenon of chemically identical molecules with different structures is coined structural polymorphism. Conventionally, structural polymorphism is observed directly by structural determination at the density map level from X-ray crystal diffraction. Although crystallography approach can report the conformation of a macromolecule with the position of each atom accurately defined in it, the exploration of structural polymorphism and interpreting biological function in terms of crystal structures is largely constrained by the crystal packing. An alternative approach to studying the macromolecule of interest in solution is thus desirable. With the advancement of instrumentation and computational methods for image analysis and reconstruction, cryo-electron microscope (cryo-EM) has been transformed to be able to produce “in solution” structures of macromolecules routinely with resolutions comparable to crystallography but without the need of crystals. Since the sample preparation of single-particle cryo-EM allows for all forms co-existing in solution to be simultaneously frozen, the image data contain rich information as to structural polymorphism. The ensemble of structure information can be subsequently disentangled through three-dimensional (3D) classification analyses. In this review, we highlight important examples of protein structural polymorphism in relation to allostery, subunit cooperativity and function plasticity recently revealed by cryo-EM analyses, and review recent developments in 3D classification algorithms including neural network/deep learning approaches that would enable cryo-EM analyese in this regard. Finally, we brief the frontier of cryo-EM structure determination of RNA molecules where resolving the structural polymorphism is at dawn.

Correlative Single-Molecule Force-FRET Measurements of DNA Hairpin Conformational Dynamics

The biological function of many macromolecules such as proteins and nucleic acids is tightly coupled to their conformation and their conformational dynamics. Therefore, studying how macromolecules fold and undergo conformational changes is crucial to understand the underlying mechanisms of biological processes. In particular DNA/RNA hairpins play essential regulatory roles in vivo, since they can regulate the interaction with nucleic acid processing proteins in processes such as transcription, recombination or replication. Therefore, understanding the principles of their structure formation is important for elucidating those biological roles. Single-molecule force spectroscopy represents an ideal tool for such studies because of its unique capability to isolate individual biomolecules and observe conformational transitions in real-time, while providing the control over the population distribution as opposed to approaches in solution. Here we report a study on FRET-labelled DNA hairpin folding transitions using high-resolution optical tweezers combined with fluorescence microscopy. We present equilibrium measurements at unprecedent trap distance stability, which allows to measure conformational state distributions over hours without altering the energy landscape. Measuring over longer time periods while keeping the trap distance within nanometer precision is crucial to capture the rare conformational states that might be overlooked in measurements over a timescale that is limited by instrumental drift. These measurements were enabled by integrating trap-distance and temperature stabilization feedback loops to achieve the ultimate stability in distance between the traps. Kinetic, energetic and structural properties of these states could be determined quantitatively from a correlative information source – force and fluorescence data, thereby providing new insights into folding pathways and energy landscape. This study shows how correlating force spectroscopy measurements with fluorescence microscopy could complement the investigation of the prevalence and energy landscape of certain conformational states with extra three-dimensional structural information of the macromolecule of interest.

Serendipitous crystallization of E. coli HPII catalase, a sequel to "the tale usually not told".

Protein crystallographers are well aware of the trap of crystallizing E. coli proteins instead of the macromolecule of interest if heterologous recombinant protein expression in E. coli was part of the experimental pipeline. Among the well-known culprits are YodA metal-binding lipocalin (25 kDa) and YadF carbonic anhydrase (a tetramer of 25 kDa subunits). We report a novel crystal form of another such culprit, E. coli HPII catalase, which is a tetrameric protein of ~340 kDa molecular weight. HPII is likely to contaminate recombinant protein samples, co-purify, and then co-crystallize with the target proteins, especially if their masses in size exclusion chromatography are ~300-400 kDa. What makes this case more interesting but also parlous, is the fact that HPII can crystallize from very low concentrations, even well below 1 mg/mL.

Valorization of whey using a biorefinery

AbstractIn Mexico, whey is discarded into drains without treatment, which represents an environmental problem. It is known that whey is a mixture of nutrients that increase biochemical demand (BOD) and chemical oxygen demand (COD), exceeding the limits established by national and international standards. Some uses of whey have been proposed to reduce the negative impact it produces on the environment. Some of these uses are the production of high‐value raw materials, separation of macromolecules of industrial interest, and the generation of energy, among others. The integral use of waste under a biorefinery scheme represents a viable alternative for the use of this waste. In this way, the chemical composition of the waste can be used to recover compounds with high added value. The main objective of this review is to propose a dairy whey biorefinery to use components of commercial interest. This biorefinery process involves three stages: the first is the separation of lactoferrin using a polyacrylamide matrix (PAM) impregnated with copper; the second is lactic fermentation using the residue resulting from the separation of lactoferrin, from which lactic acid, hydrolyzed proteins, and probiotics will be obtained. Finally, in the third stage, the residue from the previous stage can be used to generate biogas. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd

Co-Immunoprecipitation of Macrophage Migration Inhibitory Factor.

Immunoprecipitation is a technique which enables a macromolecule of interest to be isolated from heterogenous mixtures (particularly cell lysates). However, the immunoprecipitation of protein(s) can be challenging, with multiple variations of the basic technique required for successful antigenic pull-down. This depends on the target of interest, cell source, and localization. Here, immunoprecipitation of MIF from mouse and human macrophage cell lysates is described, which is both reliable and replicable, derived from multiple optimization experiments.

Interactions of polyglycerol dendrimers with human serum albumin: insights from fluorescence spectroscopy and computational modeling analysis

Polyglycerol dendrimer synthesized from glycerol core (PGLyD) is an interesting reservoir macromolecule for the design of drug delivery systems due to their adequate blood biocompatibility. However, important features as the comprehension of the structural and dynamic characteristics and the interactions of PGLyD with blood proteins receptors remain unresolved. The high affinity and transport of HSA with drugs stimulated the docking simulations utilizing PGLyD as a ligand for the main HSA docking sites IIA and IIIA. HSA and the PGLyD structures were generated with the aid of Autodock Vina and the best conformations were determined by employing molecular docking. The molecular docking results indicate a thermodynamically favorable interaction suggesting a charge transfer complex formation between HSA and PGLyD. The interaction between PGLyD and HSA was investigated by fluorescence and the quenching mechanism of fluorescence of HSA by PGLyD was discussed. The binding constants and the number of binding sites were measured. The values of thermodynamic parameters ΔG, ΔH, and ΔS were calculated at three different temperatures. The experimental and computational results suggest that hydrophobic forces play a major role in stabilizing the HSA–PGLyD complex.

The interplay of protein-ligand and water-mediated interactions shape affinity and selectivity in the LAO binding protein.

The study of binding thermodynamics is essential to understand how affinity and selectivity are acquired in molecular complexes. Periplasmic binding proteins (PBPs) are macromolecules of biotechnological interest that bind a broad number of ligands and have been used to design biosensors. The lysine-arginine-ornithine binding protein (LAO) is a PBP of 238 residues that binds the basic amino acids l-arginine and l-histidine with nm and μm affinity, respectively. It has been shown that the affinity difference for arginine and histidine binding is caused by enthalpy, this correlates with the higher number of protein-ligand contacts formed with arginine. In order to elucidate the structural bases that determine binding affinity and selectivity in LAO, the contribution of protein-ligand contacts to binding energetics was assessed. To this end, an alanine scanning of the LAO-binding site residues was performed and arginine and histidine binding were characterized by isothermal titration calorimetry and X-ray crystallography. Although unexpected enthalpy and entropy changes were observed in some mutants, thermodynamic data correlated with structural information, especially, the binding heat capacity change. We found that selectivity is conferred by several residues rather than exclusive arginine-protein interactions. Furthermore, crystallographic structures revealed that protein-ligand contributions to binding thermodynamics are highly influenced by the solvent. Finally, we found a similar backbone conformation in all the closed structures obtained, but different structures in the open state, suggesting that the binding site residues of LAO play an important role in stabilizing not only the holo conformation, but also the apo state. DATABASE: Structural data are available in the Protein Data Bank database under the accession numbers 6MLE, 6MLN, 6MLG, 6MKX, 6MLI, 6MLA, 6MKU, 6MKW, 6ML0, 6MLD, 6MLV, 6MLO, 6MLP, 6ML9, 6MLJ.

Mucin Thin Layers: A Model for Mucus-Covered Tissues.

The fate of macromolecules of biological or pharmacological interest that enter the mucus barrier is a current field of investigation. Studies of the interaction between the main constituent of mucus, mucins, and molecules involved in topical transmucoidal drug or gene delivery is a prerequisite for nanomedicine design. We studied the interaction of mucin with the bio-inspired arginine-derived amphoteric polymer d,l-ARGO7 by applying complementary techniques. Small angle X-ray scattering in bulk unveiled the formation of hundreds of nanometer-sized clusters, phase separated from the mucin mesh. Quartz microbalance with dissipation and neutron reflectometry measurements on thin mucin layers deposited on silica supports highlighted the occurrence of polymer interaction with mucin on the molecular scale. Rinsing procedures on both experimental set ups showed that interaction induces alteration of the deposited hydrogel. We succeeded in building up a new significant model for epithelial tissues covered by mucus, obtaining the deposition of a mucin layer 20 Å thick on the top of a glycolipid enriched phospholipid single membrane, suitable to be investigated by neutron reflectometry. The model is applicable to unveil the cross structural details of mucus-covered epithelia in interaction with macromolecules within the Å discreteness.

Time-dependent damping effect for the dynamics of DNA transcription

DNA is an interesting macromolecule to study because it plays a role in the body of living beings. This paper modified the Peyrard-Bishop (PB) DNA model by involving time-dependent damping. Damping on this model is considered a time-dependent perturbation. The dynamics of DNA transcription with time dependent damping effect is described by the damped nonlinear Schrodinger (DNLS) equation. Solution of the equation is obtained by applying variational method. So obtained the required parameters and done analysis using matlab. The simulation for the solution is done for nucleotide mass 5.1 × 10−25 kg, distance between base neighbor 3.4 × 10−10 m, elastic coefficient 8 N/m, depth of morse potential 0.1 eV, and width of morse potential 2 × 1010 m−1. This research showed a wave of soliton type of breather and spatial soliton. This paper also showed time-dependent damping causes changes in the amplitude and width of the DNA soliton wave.