Search Results (60)

The rate of nucleation of crystals is the subject of extensive research, since it—together with the nucleation time—determines the number of crystals growing; in turn, their number is related to their size. Experimental studies show that, for biomolecular crystals, despite the required unusually high supersaturations, the nucleation process is distinctly slow. This slowness arises from the inherent peculiarity of the nucleation of such crystals. Therefore, a prerequisite for management of the crystallization process towards the desired outcome is the molecular level understanding of the nucleation mechanism. In this paper, analyzing the mechanisms behind the nucleation process of protein crystals, it is argued that the highly inhomogeneous molecule surface is the main reason for the slow crystal nucleation: only a few small patches on their surface are capable of forming crystalline bonds. Therefore, the partner proteins must not only be brought to encounter one another but must also find each other’s binding site. In turn, this requirement imposes a severe steric restriction on the association of protein molecules, which, however, is alleviated by a rotational-diffusional reorientation. This is why particular attention is paid to this aspect of the protein crystal nucleation process. Full article

Obtaining high-resolution 3D structures of membrane proteins through X-ray crystallography remains a longstanding bottleneck in the field of structural biology. This challenge has led to the optimization of purification methods to acquire high-yielding, pure proteins suitable for crystallization. In this study, we performed crystallization screenings of purified human α4β2 nAChR using a polarized in meso method. After reconstituting the detergent-solubilized α4β2 nAChR into the LCP matrix, the samples were incubated in a polarized lipid matrix using the RMP@LMx device developed in our laboratory. The results showed that under these conditions, the α4β2-nAChR-LFC 16 complex gave a mobile fraction >0.8, suggesting that its diffusion in the polarized lipid matrix is favorable for crystal nucleation. Voltages above 70 mV restricted crystal formation due to sample dehydration. Furthermore, a lipid analysis using UPLC-ESI MS/MS revealed a profile necessary for preserving protein integrity and promoting diffusion across the LCP. We harvested a single crystal and subjected it to X-ray diffraction, resulting in reflections comparable to previous studies of the muscle-type nAChR from Torpedo californica. X-ray diffraction of a single crystal gave distinct low-resolution diffractions of protein nature. These findings lay the groundwork for further optimization of membrane protein crystallization in polarized in meso phases. Full article

Iron oxide nanoparticles (IONs) with good water dispersibility were prepared by the thermal decomposition of iron acetylacetonate (Fe(acac)₃) in the high-boiling organic solvent polyethylene glycol (PEG) using polyethyleneimine (PEI) as a modifier. The nucleation and growth processes of the crystals were separated during the reaction process by batch additions of the reaction material, which could inhibit the nucleation but maintain the crystal growth, and products with larger particle sizes and high saturation magnetization were obtained. The method of batch addition of the reactant prepared IONs with the largest particle size and the highest saturation magnetization compared with IONs reported using PEG as the reaction solvent. The IONs prepared by this method also retained good water dispersibility. Therefore, these IONs are potentially suitable for the magnetic separation of cells, proteins, or nucleic acids when large magnetic responses are needed. Full article

This research delves into the early nucleation stages of phycocyanin, a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications. The paper contrasts traditional crystallization methods with the innovative Langmuir–Blodgett nanotemplate approach, aiming to enhance molecular assembly and nucleation processes. The study employs Langmuir–Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal (SONICC) spectroscopy. This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation, capturing the intricate dynamics of protein crystallization. The experiments were conducted under controlled conditions, where surface pressure was maintained at 26 mN/m and barrier speed at 70 cm/min to optimize the monolayer formation at the air–water interface. The Langmuir–Blodgett method, compared to traditional vapor diffusion techniques, shows improvements in the uniformity and efficiency of nucleation. The sensitivity of SONICC spectroscopy significantly enhances the visualization of the nucleation process, revealing a more structured and uniform crystalline assembly in the early stages of formation. This method demonstrates a substantial improvement in nucleation dynamics, leading to a more orderly growth process and potentially larger, well-ordered crystals. Integrating Langmuir–Blodgett nanotemplates with SONICC spectroscopy offers a significant step in understanding protein crystallization processes with insights into the nucleation and growth of protein crystals and broad implications for refining crystallography methodologies of protein-based biomaterials, contributing to the advancement of structural biology and materials science. Full article

Proteins can form crystals spontaneously without crystallization experiments. These crystals can be used to determine three-dimensional structures. However, when X-ray diffraction is poor, crystal optimization is required to obtain a high-resolution crystal structure. Endo-1,4-β-xylanase from the fungus Hypocrea virens (HviGH11) spontaneously formed microcrystals after affinity purification and concentration; however, most HviGH11 microcrystals showed poor diffraction in the synchrotron X-ray and X-ray free-electron laser, so a complete three-dimensional structure could not be obtained. This study presents a method to improve the crystal quality of spontaneously grown HviGH11 microcrystals. The crystallization screening results revealed that temperature, pH, and salt were not crucial factors in increasing the solubility or preventing the spontaneous crystal growth of HviGH11. Conversely, the addition of polyethylene glycols (PEGs) as a precipitant facilitated the growth of larger HviGH11 crystals. The improved large HviGH11 crystal showed a diffraction of up to 1.95 Å when exposed to synchrotron X-rays, providing a complete three-dimensional structural dataset. Based on the nucleation rate equation, it was suggested that PEG increases the viscosity of the protein solution rather than promoting nucleation. This increase in viscosity reduced nucleation and facilitated the growth of larger HviGH11 crystals. These results provide valuable insights for future experiments aimed at increasing the size of spontaneously grown crystals. Full article

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be “ice nucleators”, potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above −30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins. Full article

Many applications in the life science and food industries require (semi-)crystalline oil-in-water (O/W) dispersions. Unfortunately, high supercooling and, thus, low temperatures are often needed to induce the crystallization of droplets. As low molecular weight emulsifiers (LMWEs) are able to act as nucleation templates, they might help to decrease the required level of supercooling. Furthermore, proteins and LMWEs are frequently co-formulated to improve the colloidal stability of emulsions and dispersions. Hence, choosing a suitable protein and LMWE mixture would allow for achieving specific product properties for controlling the solid fat content (SFC) and take advantage of the stabilization mechanisms of both emulsifiers. Therefore, this study focuses on the impact of the co-existence of β-lactoglobulin (β-lg) and phospholipids (PLs) LMWEs on the SFC of triglyceride (TAG) droplets at isothermal conditions using a thermo-optical method. When β-lg alone was used as an emulsifier, a maximum SFC of 80% was obtained at a supercooling of 32 K and 42 K for trilaurin and tripalmitin, respectively. The SFC could be increased to 100% using a PL containing saturated fatty acids (FAs) and a small hydrophilic headgroup. At the same supercooling, a PL containing saturated FAs and a large hydrophilic headgroup led to a maximum SFC of 80%. At lower supercooling, the SFC was reduced with this PL by 10% compared to β-lg alone. In addition, when the PLs had more time to adsorb and rearrange with ß-lg at the interface, even lower SFCs were observed compared to cooling directly after emulsification. Full article

Despite decades of research on fish otoliths and their capacity to serve as biochronological recorders, much remains unknown about their protein composition, the mechanisms by which proteins are incorporated into the otolith matrix, or the potential for using otolith proteins to provide insight into aspects of fish life history. We examined the protein composition of Atlantic cod (Gadus morhua) otoliths using a state-of-the-art shotgun proteomics approach with liquid chromatography coupled to an electrospray ionization-orbitrap tandem mass spectrometer. In addition to previously known otolith matrix proteins, we discovered over 2000 proteins not previously identified in cod otoliths and more than 1500 proteins not previously identified in any fish otoliths. These included three novel proteins (Somatolactin, F-actin-capping protein subunit beta, Annexin) primarily involved in binding calcium ions and likely mediating crystal nucleation. However, most of the otolith proteins were not necessarily related to otolith formation but rather to other aspects of fish physiology. For example, we identified sex-related biomarkers for males (SPATA6 protein) and females (Vitellogenin-2-like protein). We highlight some noteworthy classes of proteins having diverse functions; however, the primary goal here is not to discuss each protein separately. The number and diverse roles of the proteins discovered in the otoliths suggest that proteomics could reveal critical life history information from archived otolith collections that could be invaluable for understanding aspects of fish biology and population ecology. This proof-of-concept methodology paper provides a novel methodology whereby otolith proteomics can be further explored. Full article

The crystallization of materials from a suspension determines the structure and function of the final product, and numerous pieces of evidence have pointed out that the classical crystallization pathway may not capture the whole picture of the crystallization pathways. However, visualizing the initial nucleation and further growth of a crystal at the nanoscale has been challenging due to the difficulties of imaging individual atoms or nanoparticles during the crystallization process in solution. Recent progress in nanoscale microscopy had tackled this problem by monitoring the dynamic structural evolution of crystallization in a liquid environment. In this review, we summarized several crystallization pathways captured by the liquid-phase transmission electron microscopy technique and compared the observations with computer simulation. Apart from the classical nucleation pathway, we highlight three nonclassical pathways that are both observed in experiments and computer simulations: formation of an amorphous cluster below the critical nucleus size, nucleation of the crystalline phase from an amorphous intermediate, and transition between multiple crystalline structures before achieving the final product. Among these pathways, we also highlight the similarities and differences between the experimental results of the crystallization of single nanocrystals from atoms and the assembly of a colloidal superlattice from a large number of colloidal nanoparticles. By comparing the experimental results with computer simulations, we point out the importance of theory and simulation in developing a mechanistic approach to facilitate the understanding of the crystallization pathway in experimental systems. We also discuss the challenges and future perspectives for investigating the crystallization pathways at the nanoscale with the development of in situ nanoscale imaging techniques and potential applications to the understanding of biomineralization and protein self-assembly. Full article

Protein crystallization was first discovered in the nineteenth century and has been studied for nearly 200 years. Protein crystallization technology has recently been widely used in many fields, such as drug purification and protein structure analysis. The key to successful crystallization of proteins is the nucleation in the protein solution, which can be influenced by many factors, such as the precipitating agent, temperature, solution concentration, pH, etc., among which the role of the precipitating agent is extremely important. In this regard, we summarize the nucleation theory of protein crystallization, including classical nucleation theory, two-step nucleation theory, and heterogeneous nucleation theory. We focus on a variety of efficient heterogeneous nucleating agents and crystallization methods as well. The application of protein crystals in crystallography and biopharmaceutical fields is further discussed. Finally, the bottleneck of protein crystallization and the prospect of future technology development are reviewed. Full article

Microbially induced carbonate precipitation (MICP) is an important process in the synthesis of carbonate minerals, and thus, it is widely explored as a novel approach with potential for many technological applications. However, the processes and mechanisms involved in carbonate mineral formation in the presence of microbes are not yet fully understood. This review covers the current knowledge regarding the role of microbial cells and metabolic products (e.g., extracellular polymeric substances, proteins and amino acids) on the adsorption of divalent metals, adsorption of ionic species and as templates for crystal nucleation. Moreover, they can play a role in the mineral precipitation, size, morphology and lattice. By understanding how microbes and their metabolic products promote suitable physicochemical conditions (pH, Mg/Ca ratio and free CO₃²⁻ ions) to induce carbonate nucleation and precipitation, the manipulation of the final mineral precipitates could be a reality for (geo)biotechnological approaches. The applications and implications of biogenic carbonates in areas such as geology and engineering are presented and discussed in this review, with a major focus on biotechnology. Full article

This study aimed to explore the effects of nucleate agent sizes on lysozyme crystallization. Silica nanoparticles (SNP) with four different particle sizes of 5 nm, 15 nm, 50 nm, and 100 nm were chosen for investigation. Studies were carried out both microscopically and macroscopically. After adding SNP, the morphological defects of lysozyme crystals decreased, and the number of crystals increases with the size of the SNP. The interaction between SNP and lysozyme was further explored using UV spectroscopy, fluorescence spectroscopy, and Zeta potential. It was found that the interaction between SNP and lysozyme was mainly electrostatic interaction, which increased with the size of SNP. As a result, lysozyme could be attracted to the surface of SNP and aggregated to form the nucleus. Finally, the activity test and circular dichroism showed that SNP had little effect on protein secondary structure. Full article

Well-diffracting protein crystals are indispensable for X-ray diffraction analysis, which is still the most powerful method for structure-function studies of biomolecules. A promising approach to growing such crystals is the use of porous nucleation-inducing materials. However, while protein crystal nucleation in pores has been thoroughly considered, little attention has been paid to the subsequent growth of crystals. Although the nucleation stage is decisive, it is the subsequent growth of crystals outside the pore that determines their diffraction quality. The molecular-scale mechanism of growth of protein crystals in and outside pores is theoretically considered. Due to the low degree of metastability, the crystals that emerge from the pores grow slowly, which is a prerequisite for better diffraction. This expectation has been corroborated by experiments carried out with several types of porous material, such as bioglass (“Naomi’s Nucleant”), buckypaper, porous gold and porous silicon. Protein crystals grown with the aid of bioglass and buckypaper yield significantly better diffraction quality compared with crystals grown conventionally. In all cases, visually superior crystals are usually obtained. Our theoretical conclusion is that heterogeneous nucleation of a crystal outside the pore is an exceptional case. Rather, the protein crystals nucleating inside the pores continue growing outside them. Full article

Cyclic-olefin-copolymer (COC)-based microfluidic devices are increasingly becoming the center of highly valuable research for in situ X-ray measurements due to their compatibility with X-rays, biological compounds, chemical resistance, optical properties, low cost, and simplified handling. COC microfluidic devices present potential solutions to challenging biological applications such as protein binding, folding, nucleation, growth kinetics, and structural changes. In recent years, the techniques applied to manufacturing and handling these devices have capitalized on enormous progress toward small-scale sample probing. Here, we describe the new and innovative design aspects, fabrication, and experimental implementation of low-cost and micron-sized X-ray-compatible microfluidic sample environments that address diffusion-based crystal formation for crystallographic characterization. The devices appear fully compatible with crystal growth and subsequent X-ray diffraction experiments, resulting in remarkably low background data recording. The results highlighted in this research demonstrate how the engineered microfluidic devices allow the recording of accurate crystallographic data at room temperature and structure determination at high resolution. Full article

The classical nucleation theory shows that bulk water freezing does not occur at temperatures above ≈ −30 °C, and that at higher temperatures ice nucleation requires the presence of some ice-binding surfaces. The temperature and rate of ice nucleation depend on the size and level of complementarity between the atomic structure of these surfaces and various H-bond-rich/depleted crystal planes. In our experiments, the ice nucleation temperature was within a range from −8 °C to −15 °C for buffer and water in plastic test tubes. Upon the addition of ice-initiating substances (i.e., conventional AgI or CuO investigated here), ice appeared in a range from −3 °C to −7 °C, and in the presence of the ice-nucleating bacterium Pseudomonas syringae from −1 °C to −2 °C. The addition of an antifreeze protein inhibited the action of the tested ice-initiating agents. Full article