Crystallization Of Macromolecules Research Articles

Strong, tough and anisotropic bioinspired hydrogels.

Soft materials are widely used in tissue engineering, soft robots, wearable electronics, etc. However, it remains a challenge to fabricate soft materials, such as hydrogels, with both high strength and toughness that are comparable to biological tissues. Inspired by the anisotropic structure of biological tissues, a novel solvent-exchange-assisted wet-stretching strategy is proposed to prepare anisotropic polyvinyl alcohol (PVA) hydrogels by tuning the macromolecular chain movement and optimizing the polymer network. The reinforcing and toughening mechanisms are found to be "macromolecule crystallization and nanofibril formation". These hydrogels exhibit excellent mechanical properties, such as extremely high fracture stress (12.8 ± 0.7 MPa) and fracture strain (1719 ± 77%), excellent modulus (4.51 ± 0.76 MPa), high work of fracture (134.47 ± 9.29 MJ m-3), and fracture toughness (305.04 kJ m-2) compared with other strong hydrogels and even natural tendons. In addition, excellent conductivity, strain sensing capability, water retention, freezing resistance, swelling resistance, and biocompatibility can also be achieved. This work provides a new and effective method to fabricate multifunctional anisotropic hydrogels with high tunable strength and toughness with potential applications in the fields of regenerative medicine, flexible sensors, and soft robotics.

Synthesis and Characterization of Novel Benzimidazolium Type NHC Molecule and Its Silver Complex and Their Theoretical Analysis for Potential Anti-Cancer Activity

N-heterocyclic carbenes have initially attracted great attention of the chemists in consequence of their catalytic activity. The favorable results were also obtained in bioactivity studies for N-heterocyclic carbenes, and their transition metal complexes in following years. Theoretical methods deliver beneficial data about the several activities of molecules. One of them is the frontier orbital analysis, which supplies data about the reactivity of molecules. Another method is investigation of the molecular activity against crystals of certain macromolecules using molecular docking methods. In this study, novel 1-allyl-3-(cyclohexylmethyl)benzimidazoliumbromide and bromo[1-allyl-3-(cyclohexylmethyl)benzimidazolium-2-ylidene]silver(I) were synthesized and characterized. The characterizations were also supported by spectroscopical and theoretical methods. In order to have foresight about the bioactivities of these molecules, molecular docking methods were used to get information about the interactions between molecules and VEGFR-2, which regulates the angiogenesis, and DNA.

The effect of heat treatment of starch on its crystal structure and dynamic viscosity of starch pastes

Analysis of literature data shows the uncertainty of a number of issues of the starch dextrinization process, namely the patterns of the change in the crystal structure of starch during heat treatment and the dependence of the viscosity of starch pastes of dextrins on the conditions of starch heat treatment. These questions were chosen as the topic of this study. A decrease in the average size of starch particles by 2.7 m and an increase in the intensity of reflexes of diffractograms in the range of 2=10–200 after its heat treatment at 1350C (the temperature at which the dextrinization process starts) are well explained by dehydration and a change in the degree of crystallization of biopolymer macromolecules due to their thermal transformations. The dependence of the dynamic viscosity of hot pastes on their shear rate was studied. The steady shear flow curves showed the presence of dilatant areas as a transition from the Newtonian flow regime to the pseudoplastic flow of starch pastes. It was found that the viscosities of heat-treated starch pastes depend on the processing conditions; the viscosities increase in the following order: starch heat-treated in the presence of an acid catalyst; native starch; starch heat-treated without a catalyst for 5 hours; and starch heat-treated without a catalyst for 2.5 hours. It was proposed to divide dextrins into two separate groups: with an increased and with a reduced degree of amylose polymerization.

Solvent-Exchange-Assisted Wet Annealing: A New Strategy for Superstrong, Tough, Stretchable, and Anti-Fatigue Hydrogels.

Hydrogels are widely used in tissue engineering, soft robots, wearable electronics, etc. However, it remains a great challenge to develop hydrogels possessing simultaneously high strength, large stretchability, great fracture energy, and good fatigue threshold to suit different applications. Herein, a novel solvent-exchange-assisted wet-annealing strategy is proposed to prepare high performance poly(vinyl alcohol) hydrogels by extensively tuning the macromolecular chain movement and optimizing the polymer network. The reinforcing and toughening mechanisms are found to be "macromolecule crystallization and entanglement". These hydrogels have large tensile strengths up to 11.19 ± 0.27MPa and extremely high fracture strains of 1879 ± 10%. In addition, the fracture energy and fatigue threshold can reach as high as 25.39 ± 6.64kJ m-2 and ≈1233 J m-2 , respectively. These superb mechanical properties compare favorably to those of other tough hydrogels, organogels, and even natural tendons and synthetic rubbers. This work provides a new and effective method to fabricate superstrong, tough, stretchable, and anti-fatigue hydrogels with potential applications in artificial tendons and ligaments.

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive.

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physio-logical temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system - the CFEL TapeDrive - with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.

Formation of UHMWPE Nanofibers during Solid-State Deformation.

A network of nanofibers is formed in situ through solid-state deformation of disentangled ultra-high molecular weight polyethylene (dis-UHMWPE) during compounding with a polyolefin elastomer below the melting temperature of dis-UHMWPE crystals. Dis-UHMWPE was prepared in the form of powder particles larger than 50 μm by polymerization at low temperatures, which favored the crystallization and prevention of macromolecules from entangling. Shearing the blend for different durations and at different temperatures affects the extent to which the grains of dis-UHMWPE powder deform into nanofibers. Disentangled powder particles could deform into a network of nanofibers with diameters between 110 and 340 nm. The nanocomposite can be further sheared for a longer time to decrease the diameter of dis-UHMWPE nanofibers below 40 nm, being still composed of ≈70 wt.% of crystalline and ≈30 wt.% of amorphous components. Subsequently, these thinner fibers begin to melt and fragment because they are thinner and also because the amorphous defects locally decrease the nanofibers' melting temperature, which results in their fragmentation and partial loss of nanofibers. These phenomena limit the thickness of dis-UHMWPE nanofibers, and this explains why prolonged or more intense shearing does not lead to thinner nanofibers of dis-UHMWPE when compounded in a polymeric matrix.

Crystallization of Nano-Sized Macromolecules by the Example of Hexakis-[4-{(N-Allylimino)methyl}phenoxy]cyclotriphosphazene.

The synthesized compound was characterized by 31P, 13C, and 1H NMR spectroscopy and MALDI-TOF mass spectroscopy. According to DSC data, the compound was initially crystalline, but the crystal structure was defective. The crystals suitable for X-ray diffraction study were prepared by slow precipitation of the compound from a solution by a vapor of another solvent. A study of the single crystal obtained in this way demonstrated that the phosphazene ring has a flattened chair conformation. It was found that the sphere circumscribed around the compound molecule has a diameter of 2.382 nm.

Screw dislocation-induced pyramidal crystallization of dendron-like macromolecules featuring asymmetric geometry.

We report herein that dendron-shaped macromolecules ABn crystallize into well-ordered pyramid-like structures from mixed solvents, instead of spherical motifs with curved structures, as found in the bulk. The design of the asymmetric molecular architecture and the choice of mixed solvents are applied as strategies to manipulate the crystallization process. In mixed solvents, the solvent selection for the Janus macromolecule and the existence of dominant crystalline clusters contribute to the formation of flat nanosheets. Whereas during solvent evaporation, the bulkiness of the asymmetric macromolecules easily creates defects within 2D nanosheets which lead to their spiral growth through screw dislocation. The size of the nanosheets and the growth into 2D nanosheets or 3D pyramidal structures can be regulated by the solvent ratio and solvent compositions. Moreover, macromolecules of higher asymmetry generate polycrystals of lower orderliness, probably due to higher localized stress.

Two-Step Mechanism of Macromolecular Nucleation and Crystallization: Field Theory and Simulations

We developed a phase-field method of simulations of crystallization of macromolecules from solutions, which provides a good combination of the efficiency, thermodynamic consistency, and predictive ...

Optimization of crystallization of biological macromolecules using dialysis combined with temperature control.

A rational way to find the appropriate conditions to grow crystal samples for bio-crystallography is to determine the crystallization phase diagram, which allows precise control of the parameters affecting the crystal growth process. First, the nucleation is induced at supersaturated conditions close to the solubility boundary between the nucleation and metastable regions. Then, crystal growth is further achieved in the metastable zone - which is the optimal location for slow and ordered crystal expansion - by modulation of specific physical parameters. Recently, a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature-precipitant-concentration phase diagrams has been constructed. Here, it is demonstrated that a thorough knowledge of the phase diagram is vital in any crystallization experiment. The relevance of the selection of the starting position and the kinetic pathway undertaken in controlling most of the final properties of the synthesized crystals is shown. The rational crystallization optimization strategies developed and presented here allow tailoring of crystal size and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination.

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

A comparative anatomy of protein crystals: lessons from the automatic processing of 56 000 samples.

The fully automatic processing of crystals of macromolecules has presented a unique opportunity to gather information on the samples that is not usually recorded. This has proved invaluable in improving sample-location, characterization and data-collection algorithms. After operating for four years, MASSIF-1 has now processed over 56 000 samples, gathering information at each stage, from the volume of the crystal to the unit-cell dimensions, the space group, the quality of the data collected and the reasoning behind the decisions made in data collection. This provides an unprecedented opportunity to analyse these data together, providing a detailed landscape of macromolecular crystals, intimate details of their contents and, importantly, how the two are related. The data show that mosaic spread is unrelated to the size or shape of crystals and demonstrate experimentally that diffraction intensities scale in proportion to crystal volume and molecular weight. It is also shown that crystal volume scales inversely with molecular weight. The results set the scene for the development of X-ray crystallography in a changing environment for structural biology.

Temperature-induced nucleation and growth of protein single crystals

Two techniques for the nucleation and growth of single crystals of biological macromolecules are proposed. The first one utilizes a very slow temperature shift at a capillary point where the crystal is to be grown. This allows to suppress an undesirable multiple nucleation. The second technique includes several local rapid temperature changes (a temperature “shock”) forcing the nucleation at the given point. These techniques were successfully tested while growing single crystals of lysozyme and xylanase respectively.

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules.

High-brilliance X-ray beams coupled with automation haveled to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

Applications of gradient magnetic fields in studies of biological macromolecules

As a kind of physical environment, magnetic fields are widely used in many different fields, ranging from scientific research to industrial production. With the rapid development of magnetic technology, the importance of magnetic fields in scientific research and practical applications has become increasingly prominent. One notable research field is the application of magnetic fields in the research of biological macromolecules. In this research field, gradient magnetic fields, as one kind of magnetic fields, have attracted much attention and have been widely used in various research fields because they can induce effects caused not only by a magnetic field but also by a magnetic field gradient. This feature makes magnetic fields applicable in many research areas, except for those directly related to the magnetic field itself. So far, the application-based research of gradient magnetic fields involving biological macromolecules has mainly focused on the crystallization, separation and purification as well as the self-assembly of biological macromolecules. Since biological macromolecules usually need to complete various processes in their solution state, the influence of gradient magnetic fields on biological macromolecules is mainly realized through their influence on the solution system. It has been found that a gradient magnetic field can affect convection in biological macromolecular solutions through Lorentz forces and magnetization forces; in practice, partial or even complete suppression of convection can often be observed. In addition, other conditions or parameters of solutions are significantly affected by the gradient magnetic field. For example, in a gradient magnetic field, the following phenomena may occur: (1) superparamagnetic particles in the solution are strongly attracted by the magnetization force, and quick clustering of these particles can happen; (2) the solution itself may have some kind of solution structure; (3) the aggregation of biological macromolecules may show a preferred orientation in the magnetic field direction; (4) the diffusion coefficient of biological macromolecules in the solution will decrease; (5) the solutions viscosity will increase, and so on. As a result of these effects, the following phenomena can be observed: during the crystallization of biological macromolecules, a preferred orientation of the crystals and crystal quality improvement of the biological macromolecules can happen; in the process of separation and purification of biological macromolecules, high-purity and highly bioactive macromolecules can be obtained rapidly and efficiently; and, in the process of the self-assembly of biological macromolecules, abnormal behaviours can occur. Based on these specific effects, the utilization of gradient magnetic fields can be further explored in more professional applications. Currently, the most common applications include the utilization of gradient magnetic fields for obtaining high- quality protein crystals and the achievement of highly efficient and low-cost separation and purification of biological macromolecules. It is obvious that gradient magnetic fields have very important application value in biological macromolecular structure analysis technology, biological drug preparation and preservation technology, theoretical studies involving biomolecular phase separation and nucleation processes, and many other directions. In this paper, the fundamental effects of the gradient magnetic field on the solution of biological macromolecules are reviewed, the applications based on the effects are discussed, and the future prospects in this field are anticipated.

A Dimension Reduction Method for cryo-EM Image Analysis

In the field of structural biology, X-ray crystallography and NMR spectroscopy have been two major high-resolution techniques. The former requires crystallization of macromolecules while the latter is a solution method but would meet limit when the macromolecule is large. Very recently, breakthrough in the camera together with microscopy automation, advancement of algorithm and GPU-accelerated computations has transformed cryo-EM to become a standard technique to solve structures of macromolecules at near atomic resolution. In contrast to X-ray crystallography, single particle cryo-EM does not need crystalline sample because it focuses the transmitted electrons to directly provide electron density information of a macromolecule. As such, atomic structures of several difficult macromolecules not amenable to X-ray crystallography have now been rapidly revealed by single particle cryo-EM and single particle cryo-EM has been chosen as the method of 2015 by Nature Method. Because biological molecules are extremely vulnerable, very low doses of electrons are used by cryo-EM, leading to very noisy images. Here, we propose a dimension reduction method 2SDR to a set of single-particle cryo-electron-microscopy (cryo-EM) images and demonstrated that 2SDR can realize effective de-noising.

Structural Basis of Enhanced Crystallizability Induced by a Molecular Chaperone for Antibody Antigen-Binding Fragments

Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody–antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (VHH) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody–ligand complex and lowers its aggregation temperature to favor crystallization. The VHH crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules.

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal.

Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 µm3, i.e. no more than 6 × 105 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 × 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.

MPI tray: a versatile crystallization plate for membrane proteins

High-throughput crystallization of biological macromolecules is usually performed on multi-well plates, the design of which needs to address different and sometimes conflicting requirements. In this regard, handling of membrane proteins presents a particular challenge owing to the common use of detergents with associated effects on surface tension. Reported here is the design of a new crystallization plate, termed the MPI tray, which is optimized for UV and visible imaging with membrane protein samples. Following basic considerations regarding geometry and material, the surface properties of the plate were subjected to extensive analysis and modification in order to improve the performance in a robotic environment. An electrostatic surface potential was identified as the major problem affecting the automated setup of experiments, and it was found that treatment of the crystallization plate with ethanol is effective in removing this potential.