Search Results (80)

Dermatophytosis is a fungal infection that affects the skin, hair, and nails, impacting approximately 25% of the global population. Nannizzia gypsea is a geophilic fungus that can cause infections in humans and animals. Several studies have been conducted regarding its virulence, or ability to cause disease. This species may produce keratinolytic enzymes and form biofilms, which can increase resistance to treatment. Thus, this study focuses on investigating the biofilm formation of N. gypsea isolated from canine dermatophytosis using an ex vivo hair model, its biofilm extracellular matrix macromolecular contents, and the expression of genes involved in the colonization of keratinized surfaces. The biofilm was analyzed for metabolic activity using the XTT reduction assay, crystal violet staining to measure biofilm biomass, scanning electron microscopy (SEM), and the presence of polysaccharides, proteins, and extracellular DNA in the biofilm extracellular matrix. The virulence genes subtilisin 7, fungalysin (extracellular metalloproteinase), and efflux pump (Multidrug and Toxin Extrusion Protein 2) were evaluated by qPCR, comparing the planktonic and biofilm phenotypes. N. gypsea formed a robust biofilm, which matured after 5 days. Scanning electron microscopy (SEM) revealed the presence of an extensive extracellular matrix. In the hair model, the characteristic ectothrix parasitism of the species is observable. The gene expression analysis revealed a higher expression of all evaluated genes in the biofilm form compared to the planktonic form. Thus, N. gypsea exhibits a biofilm characterized by a robust extracellular matrix and high gene expression of factors related to pathogenesis and resistance. Full article

The properties of compatibilized blends of polyethylene (PE) and polypropylene (PP), having reduced macromolecular entanglements, were studied. The density of PP macromolecular entanglements was controlled by prior disentangling in solution. The polymer ratio in the blend was 4:1 or 1:4. An ethylene–octene copolymer was used as a compatibilizer. The melt blending process resulted in good dispersion of the minority component, with slightly larger inclusions when more disentangled PP was used. Rheological studies confirmed the achievement of different entanglement densities of PP macromolecules in the blends. The partial disentanglement did not affect the thermal stability of the blends. During the isothermal crystallization studies, faster growth of PP spherulites was observed in the blend with reduced entanglements, which also influenced the entire crystallization process. The recovery time of equilibrium entanglement was investigated and it turned out to be 45 min if the blend was annealed at 190 °C, which was shorter than in the analogous homopolymer. Studies of tensile properties showed that in blends with a majority share of polyethylene, the elongation at break increased with the disentanglement of the minority component, due to better bonding of the blend components and thus the reduction in microcavitation. Full article

In this study, a novel macromolecular flame retardant (MFR) with a phosphine oxide structure is successfully synthesized to improve the flame retardancy of polyamide 6 (PA6). Following this, the flame-retardant polyamide 6 (FR–PA6) is prepared via melt blending the MFR with PA6. Results indicate that the introduction of MFR has little effect on the melting and crystallization temperature of FR–PA6. While it slightly reduces the thermal stability of PA6, MFR significantly enhances its flame retardancy. The limiting oxygen index of FR–PA6 increases from 21.8% to 28.2%, and it successfully passes the UL-94 V-0 rating when it contains 0.5 wt% of phosphorus. Compared with pure PA6, the av-EHC of FR–PA6 is reduced by 32.2% and the SEA is increased by 66.7%. The MFR showed a flame-retardant mechanism in both the gas phase and the condensed phase. In the gas phase, the decomposition of MFR releases phosphorus-containing free radicals to interrupt the combustion chain reaction and reduces the concentration of the combustible caprolactam. In the condensed phase, the MFR promotes faster formation of melt droplets during combustion, taking heat away from the burning PA6 timely. Full article

Lysozyme plays a crucial role in the natural immune system, protecting against invading bacteria or viruses. The room-temperature (RT) structure of lysozymes is important for understanding accurate structural information compared to the crystal structure determined at cryogenic temperature. Several RT structures of lysozymes are determined by serial crystallography, but their temperature-dependent structural properties are not fully elucidated. To better understand the temperature-dependent structural change, the RT and cryogenic temperature structures of hen egg white lysozyme (HEWL) were determined by serial synchrotron crystallography (SSX) and macromolecular crystallography (MX), respectively. Structural comparisons of HEWL^RT and HEWL^Cryo showed that the positions of the loops above the substrate-binding cleft of HEWL differed. The width of the substrate-binding cleft between the α- and β-domains of HEWL^RT was wider than that of HEWL^Cryo. The distance between the two catalytic residues Glu53 and Asp70 and their interaction with neighbor residues and water molecules showed the distant between HEWL^RT and HEWL^Cryo. Due to temperature, the subtle movements of the active site and substrate-binding cleft of HEWL led to different docking results for N-acetylglucosamine and N,N′,N″-triacetylchitotriose. These results will provide useful information to more accurately understand the molecular function of HEWL and insights into the temperature effects for ligand design. Full article

Serial crystallography (SX) enables macromolecular structure determination at biologically relevant temperatures while minimizing radiation damage. This technique relies on processing numerous diffraction images from multiple crystals to construct a complete dataset for three-dimensional structure determination. Although increasing the volume of SX diffraction data improves data quality, excessive data collection reduces beamtime efficiency. Therefore, understanding the relationship between data volume and data quality is crucial for the efficient use of SX beamtime. In this study, serial synchrotron crystallography datasets from lysozyme and glucose isomerase were analyzed to assess the impact of varying diffraction data volumes on processing statistics and structural determination outcomes. Data processing statistics and structure refinement metrics improved as the volume of integrated diffraction data increased; however, the rate of improvement in data quality was not proportional to the number of integrated diffraction patterns. Furthermore, the rate of improvement in data processing statistics decreased beyond a certain threshold volume. These findings expand our understanding of SX data processing and provide insights into optimizing the efficiency of data processing. Full article

Microfluidics provides cutting-edge technological advancements for the in-channel manipulation and analysis of dissolved macromolecular species. The intrinsic potential of microfluidic devices to control key characteristics of polymer macromolecules such as their size distribution requires unleashing its full capacity. This work proposes a combined approach to analyzing the microscale behavior of polymer solutions and modifying their properties. We utilized the idea of modeling cross-channel diffusion in polydisperse polymer microflows using dynamic light scattering size distribution curves as the source data. The model was implemented into a Matlab script which predicts changes in polymer size distribution at microfluidic chip outputs. We verified the modeling predictions in experiments with a series of microchips by detecting the optical responses of injected nematic liquid crystals in the presence of microfluidic polymer species and analyzing the polymer size distribution after microfluidic processing. The results offer new approaches to tuning the size and dispersity of macromolecules in solution, developing auxiliary tools for such techniques as dynamic light scattering, and labs-on-chips for the combined diagnostics and processing of polymers. Full article

In macromolecular crystallography (MX), a complete diffraction dataset is essential for determining the three-dimensional structure. However, collecting a complete experimental dataset using a single crystal is frequently unsuccessful due to poor crystal quality or radiation damage, resulting in the collection of multiple incomplete datasets. This issue can be solved by merging incomplete diffraction datasets to generate a complete dataset. This study introduced a new approach for merging incomplete datasets from MX to generate a complete dataset using serial crystallography (SX). Six incomplete diffraction datasets of β-glucosidase from Thermoanaerobacterium saccharolyticum (TsaBgl) were processed using CrystFEL, an SX program. The statistics of the merged data, such as completeness, CC, CC*, R_split, R_work, and R_free, demonstrated a complete dataset, indicating improved quality compared with the incomplete datasets and enabling structural determination. Also, the merging of the incomplete datasets was processed using four different indexing algorithms, and their statistics were compared. In conclusion, this approach for generating a complete dataset using SX will provide a new opportunity for determining the crystal structure of macromolecules using multiple incomplete MX datasets. Full article

Hydrogen is the lightest atom and composes approximately half of the atomic content in macromolecules, yet their location can only be inferred or predicted in most macromolecular structures. This is because hydrogen can rarely be directly observed by the most common structure determination techniques (such as X-ray crystallography and electron cryomicroscopy). However, knowledge of hydrogen atom positions, especially for enzymes, can reveal protonation states of titratable active site residues, hydrogen bonding patterns, and the orientation of water molecules. Though we know they are present, this vital layer of information, which can inform a myriad of biological processes, is frustratingly invisible to us. The good news is that, even at modest resolution, neutron crystallography (NC) can reveal this layer and has emerged this century as a powerful tool to elucidate enzyme catalytic mechanisms. Due to its strong and coherent scattering of neutrons, incorporation of deuterium into the protein crystal amplifies the power of NC. This is especially true when solvation and the specific participation of key water molecules are crucial for catalysis. Neutron data allow the modeling of all three atoms in water molecules and have even revealed previously unobserved and unique species such as hydronium (D₃O⁺) and deuteroxide (OD⁻) ions as well as lone deuterons (D⁺). Herein, we briefly review why neutrons are ideal probes for identifying catalytically important water molecules and these unique water-like species, limitations in interpretation, and four vignettes of enzyme success stories from disparate research groups. One of these groups was that of Dr. Chris G. Dealwis, who died unexpectedly in 2022. As a memorial appreciation of his scientific career, we will also highlight his interest and contributions to the neutron crystallography field. As both the authors were mentored by Chris, we feel we have a unique perspective on his love of molecular structure and admiration for neutrons as a tool to query those structures. Full article

Radiation damage is an inherent problem in macromolecular crystallography because it impairs the diffraction quality of crystals and produces inaccurate structural information. Understanding radiation damage in protein structures is crucial for accurate structural interpretation and effective data collection. This study undertook X-ray data collection and structure determination of thermolysin (TLN), which contains Zn and Ca ions, by using three different X-ray doses to improve our understanding of the radiation damage phenomena on metal ions in proteins. Data processing revealed typical global radiation damage in TLN, such as an increase in unit cell volume, R_merge value, and Wilson B-factor. An analysis of the B-factor indicated that radiation damage at the Zn and Ca sites in TLN increased with higher X-ray doses. However, the distance between the metal ions and their interacting residues in TLN was not significantly affected, suggesting that radiation damage to the metal ions has a minimal effect on these interactions. Moreover, the increase in the B-factor of the metal ions according to the X-ray dose was similar to that in the B-factor of the residues interacting with the metal ions. These results expand our understanding of radiation damage phenomena in macromolecules and can be used to improve data collection strategies. Full article

Human serum albumin (HSA) is an endogenous inhibitor of angiotensin I-converting enzyme (ACE) and, thus, plays a key role in the renin–angiotensin–aldosterone system (RAAS). However, little is known about the mechanism of interaction between these proteins, and the structure of the HSA–ACE complex has not yet been obtained experimentally. The purpose of the presented work is to apply computer modeling methods to study the interaction of HSA with ACE in order to obtain preliminary details about the mechanism of their interaction. Ten possible HSA–ACE complexes were obtained by the procedure of macromolecular docking. Based on the number of steric and polar contacts between the proteins, three leading complexes were selected, the stabilities of which were then tested by molecular dynamics (MD) simulation. Based on the results of MD simulation, the two most probable conformations of the HSA–ACE complex were selected. The analysis of these conformations revealed that the processes of oxidation of the thiol group of Cys34 of HSA and the binding of albumin to ACE can reciprocally affect each other. Known point mutations in the albumin molecules Glu82Lys, Arg114Gly, Glu505Lys, Glu565Lys and Lys573Glu can also affect the interaction with ACE. According to the result of MD simulation, the known ACE mutations, albeit associated with various diseases, do not affect the HSA–ACE interaction. A comparative analysis was performed of the resulting HSA–ACE complexes with those obtained by AlphaFold 3 as well as with the crystal structure of the HSA and the neonatal Fc receptor (FcRn) complex. It was found that domains DI and DIII of albumin are involved in binding both ACE and FcRn. The obtained results of molecular modeling outline the direction for further study of the mechanisms of HSA–ACE interaction in vitro. Information about these mechanisms will help in the design and improvement of pharmacotherapy aimed at modulation of the physiological activity of ACE. Full article

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI’s unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased V_max with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells. Full article

Knowledge of hydrogen locations and protonation states is critical for a fundamental understanding of biological macromolecular function/interactions, and neutron macromolecular crystallography (NMX) is uniquely suited among the experimental structural-determination methods to provide this information. However, despite its potential, NMX remains a relatively niche technique, due to substantial limitations. This review explores NMX’s role amongst the evolving landscape of structural biology, comparing and contrasting it to the historical gold standard of X-ray macromolecular crystallography (X-ray MX) and the increasingly prevalent electron-based methods—i.e., electron microscopy (EM) and electron diffraction (ED). Forthcoming developments (e.g., the European Spallation Source in Lund, Sweden, coming online) are expected to substantially address current limitations and ensure NMX will remain relevant in the coming decades. Full article

Temperature directly influences the function and structure of proteins. Crystal structures determined at room temperature offer more biologically relevant structural information regarding flexibility, rigidity, and thermal motion than those determined by conventional cryocrystallography. Crystal structures can be determined at room temperature using conventional macromolecular crystallography (MX) or serial crystallography (SX) techniques. Among these, MX may theoretically be affected by radiation damage or X-ray heating, potentially resulting in differences between the room temperature structures determined by MX and SX, but this has not been fully elucidated. In this study, the room temperature structure of xylanase GH11 from Thermoanaerobacterium saccharolyticum was determined by MX (RT-TsaGH11-MX). The RT-TsaGH11-MX exhibited both the open and closed conformations of the substrate-binding cleft within the β-sandwich fold. The RT-TsaGH11-MX showed distinct structural changes and molecular flexibility when compared with the RT-TsaGH11 determined via serial synchrotron crystallography. The notable molecular conformation and flexibility of the RT-TsaGH11-MX may be induced by radiation damage and X-ray heating. These findings will broaden our understanding of the potential limitations of room temperature structures determined by MX. Full article

To resolve photons hungry for weak diffraction samples by the crystallographic method, a double-multilayer monochromator (DMM) was employed on an undulator beamline (BL17UM) at the Shanghai Synchrotron Radiation Facility (SSRF) to provide a focused sub-micron beam with high brightness for macromolecular crystallography experiments. High-quality crystallographic datasets from model protein crystal samples were collected and processed by an existing crystallographic program for structure solution and refinement. The data quality was compared with datasets from a normal silicon crystal monochromator to evaluate the bandwidth of the DMM effect on these crystallographic data. This experiment demonstrates that multilayer optics on an undulator beamline may play a valuable role in satisfying the demands of structure-related research, which requires novel methods. Full article

Radiation damage is an inherent challenge in macromolecular crystallography (MX). This diminishes the diffraction quality and also compromises the accuracy of the crystal structure. Investigating the impact of radiation damage on the crystal quality and structure can offer valuable insights into the structural interpretation and data collection strategy. Selenomethionine (SeMet, Mse) is an amino acid that exists in nature and contains a high-Z atom, i.e., selenium (Se), which is sensitive to radiation damage; however, little is known regarding the radiation damage of this amino acid. To better understand the radiation damage that affects SeMet, we investigated the radiation damage to a SeMet-substituted substrate-binding protein from Rhodothermus marinus. As the X-ray dose increased, the quality of the data statistics deteriorated. In particular, an increase in the X-ray dose increased the negative Fo-Fc electron density map near the Se atom of the Mse residue, while no negative Fo-Fc electron density map was observed in the other atoms (O, C, and N). Radiation damage increased the absolute B-factor value of the Se atom in the Mse residue, which was higher than that of the other atoms. This indicates that Se is more sensitive to radiation damage than other atoms. These results will contribute to advancing our knowledge of the radiation damage that can occur in MX. Full article