Protein Crystal Growth Research Articles

Two-step crystallization in a supersaturated solution with application to protein crystal growth: Theory and analytical solutions

A theory of two-step nucleation and evolution of crystals with allowance for their diffusion in the space of particle radii is developed. The theory is based on a two-step growth law of crystals appearing in dense liquid precursors, initially evolving within them with a diffusion-limited growth law, and then leaving them and evolving with another growth law. This two-step crystal growth law influences the integrodifferential system of kinetic and balance equations for the crystal-size distribution function and liquid supersaturation. The system of integrodifferential equations for the evolution of polydisperse ensemble of crystals is solved using the integral Laplace transform and saddle-point methods for the Weber–Volmer–Frenkel–Zel’dovich and Meirs kinetic mechanisms. A complete analytical solution to this non-linear system has been constructed in a parametric form. Namely, the particle-size distribution function, liquid supersaturation and time have been found as the functions of decision variable — the maximum size of crystals. The theory is in agreement with the experimental data on the growth of beta-lactoglobulin and insulin crystals.

Insights into Early Phases of Phycocyanin Crystal Formation via SONICC Spectroscopy

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.

Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Determining Protein Structures Using X-Ray Crystallography.

X-ray crystallography is a robust and widely used technique that facilitates the three-dimensional structure determination of proteins at an atomic scale. This methodology entails the growth of protein crystals under controlled conditions followed by their exposure to X-ray beams and the subsequent analysis of the resulting diffraction patterns via computational tools to determine the three-dimensional architecture of the protein. However, achieving high-resolution structures through X-ray crystallography can be quite challenging due to complexities associated with protein purity, crystallization efficiency, and crystal quality.In this chapter, we provide a detailed overview of the gene to structure determination pipeline used in X-ray crystallography, a crucial tool for understanding protein structures. The chapter covers the steps in protein crystallization, along with the processes of data collection, processing, structure determination, and refinement. The most commonly faced challenges throughout this procedure are also addressed. Finally, the importance of standardized protocols for reproducibility and accuracy is emphasized, as they are crucial for advancing the understanding of protein structure and function.

The regulation of protein crystallization using choline chloride precipitant and its mechanism

Inorganic salts are the most commonly used precipitants in protein crystallization. However, due to their strong ability to destroy the hydration shell and diffuse electric double layer of proteins, the use of inorganic salts can easily cause protein precipitation. This work studied protein crystallization using organic salt choline chloride (cc) as the precipitant. Compared with NaCl, crystallization, it was found that lysozyme could nucleate at a lower supersaturation and the single-crystal area was significantly enlarged with cc. Even at a high supersaturation of nearly 30, lysozyme crystals could still maintain a complete tetragonal morphology with no urchin-like crystals in cc solution. Furthermore, the average crystal size in cc solution was larger than that in NaCl solution even at a lower supersaturation. In addition, the quantitative efficiency of yield was about equal in cc and NaCl system. Combining Zeta potential, fluorescence spectrum and MD simulation, it was found that cc could interact with lysozyme through hydrogen bond, which weakened the damage of Cl− to diffuse electric double layer of proteins and increased the stability of lysozyme. DLS revealed that the presence of cc could promote the formation of large protein aggregates, which was favorable for crystal growth. In addition, CD spectra and lysozyme activity showed that the presence of cc did not affect the secondary structure and activity of lysozyme. This work proved that cc could be used as a substitute precipitant to grow protein crystals with large size and improve the stability of protein crystallization.

Protein crystal growth under microgravity

Protein crystal growth under microgravity

Preparation and Characterization of Metal-Organic Framework Coatings for Improving Protein Crystallization Screening.

Modifying crystallization plates can significantly impact the success rate and quality of protein crystal growth, making it a helpful strategy in protein crystallography. However, appropriate methods for preparing nano-sized particles with a high specific surface area and strategies for applying these nanoparticles to form suitable coatings on crystallization plate surfaces still need to be clarified. Here, we utilized both an ultrasonic crusher and a high-pressure homogenizer to create a nano metal-organic framework (MOF), specifically HKUST-1, and introduced a solvent evaporation method for producing MOF coatings on 96-well crystallization plates to induce protein crystal growth. The morphology of MOF coatings on the resin surface of the plate well was characterized using optical and scanning electron microscopy. Compared to the control group, crystallization screening experiments on nine proteins confirmed the effectiveness of plates with MOF coatings. Applying MOF coatings to crystallization plates is an easy-to-use, time-efficient, and potent tool for initiating crystallization experiments.

Taylor vortex-based protein crystal nucleation enhancement and growth evaluation in batchwise and slug flow crystallizers

The utilisation of nucleation seeds, coupling nucleation and growth during crystallization and realistic continuum are currently relatively robust, reliable and scalable methods for protein crystallization optimization. Here, we design a segmented crystallization method, in which combines the characteristics of the Taylor vortex enhanced lysozyme nucleation stage. Different crystallizer are used for the growth segment so that the nucleation segment and the growth segment are organically combined during lysozyme crystallization. During the crystal growth stage, the crystallization properties of the Couette-Taylor (CT) crystallizer, mixing Tank (MT) crystallizer and tubular crystallizer were evaluated. The experimental results emphasize the importance of shear rate, temperature, and fluid velocity in the design of segment crystallization methods to obtain products with complete crystal morphology and uniform size distribution. This strategy successfully expanded the lysozyme crystallization from the original 10 mL scale to 200 mL, realized the continuous process, and providing a promising strategy for the design and operation of continuous SFC to produce high value protein crystals in the future.

X-ray Diffraction Topography (Imaging) of Crystals Grown from Solution: A Short Review

A short review of X-ray topographic studies of crystals grown from solution is presented. The dislocation configurations characteristic of such crystals are described, and Klapper’s explanation in terms of minimization of the elastic energy of a dislocation per unit growth length is highlighted. It is shown that the principles apply to crystals grown not only from low temperature solution but also by hydrothermal growth and growth from fluxed melts. Recent studies of growth of protein crystals have found that they have similar dislocation configurations and characteristics to those of ionic and small organic molecule crystals grown from solution.

Laser ablation combined with Nanoimprint Lithography technology as new surface engineering approach to produce novel polymer-based heteronucleants for recalcitrant protein crystallization

Macromolecule crystallography is currently the most powerful technique for determining the three dimensional structure of proteins at atomic resolution. However, despite its enormous level of technical development achieved, it is still limited by the great difficulty of obtaining crystals that, after exposure to X-rays, allow a high-resolution diffraction pattern from which the structure of the target protein can be elucidated. Even today, the crystallization process is a trial-and-error procedure that requires enormous experimental effort and high economic costs. In this research work, Laser Ablation technology in combination with NanoImprint Lithography was applied on Polycarbonate surfaces to produce novel heteronucleating agents containing multiscale surface topographies (in the micro- and submicro-scale) that promote the growth of protein crystals. Two human proteins, known to be recalcitrant to crystallization when using standard protocols, were used in the assays: (i) a construct of the CNNM4 magnesium transporter lacking amino acid residues 545–730 (HsCNNM4545-730) and (ii) cystathionine beta-synthase enzyme lacking residues 516–525 (HsCBSΔ516-525). The surface engineering solution was validated by comparing the number of novel crystallization conditions induced by those surfaces compared against the control and the use of the Naomi’s Nucleant, a commercially available heteronucleant made of mesoporous bioglass. The aspect ratio of the topography emerges as the topographical parameter that most contributes to protein growth on patterned surfaces. Additionally, the presence of a random distribution of micropores as well as a hierarchical (merging micro- and nano - scale features) surface topography also resulted in a more efficient crystallization process, showing an increment of between 38 and 68% in the number of new crystallization conditions, depending on the surface topography and protein selected. A conclusion is made that Laser Ablation with ultrashort pulses and the combination with Nanoimprint Lithography can be efficiently applied on polymeric substrates to produce a novel concept of heteronucleants that would bridge the gap to the development of universal nucleation agents.

Protein Crystals Nucleated and Grown by Means of Porous Materials Display Improved X-ray Diffraction Quality.

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.

Lysozyme crystallization in hydrogel media under ultrasound irradiation

Sonocrystallization implies the application of ultrasound radiation to control the nucleation and crystal growth depending on the actuation time and intensity. Its application allows to induce nucleation at lower supersaturations than required under standard conditions. Although extended in inorganic and organic crystallization, it has been scarcely explored in protein crystallization. Now, that industrial protein crystallization is gaining momentum, the interest on new ways to control protein nucleation and crystal growth is advancing. In this work we present the development of a novel ultrasound bioreactor to study its influence on protein crystallization in agarose gel. Gel media minimize convention currents and sedimentation, favoring a more homogeneous and stable conditions to study the effect of an externally generated low energy ultrasonic irradiation on protein crystallization avoiding other undesired effects such as temperature increase, introduction of surfaces which induce nucleation, destructive cavitation phenomena, etc. In-depth statistical analysis of the results has shown that the impact of ultrasound in gel media on crystal size populations are statistically significant and reproducible.

Existence of twisting in dislocation-free protein single crystals

The growth of high-quality protein crystals is a prerequisite for the structure analysis of proteins by X-ray diffraction. However, dislocation-free perfect crystals such as silicon and diamond have been so far limited to only two kinds of protein crystals, such as glucose isomerase and ferritin crystals. It is expected that many other high-quality or dislocation-free protein crystals still exhibit some imperfection. The clarification of the cause of imperfection is essential for the improvement of crystallinity. Here, we explore twisting as a cause of the imperfection in high-quality protein crystals of hen egg-white lysozyme crystals with polymorphisms (different crystal forms) by digital X-ray topography with synchrotron radiation. The magnitude of the observed twisting is 10−6 to 10−5°/μm which is more than two orders smaller than 10−3 to 104°/μm in other twisted crystals owing to technique limitations with optical and electron microscopy. Twisting is clearly observed in small crystals or in the initial stage of crystal growth. It is uniformly relaxed with crystal growth and becomes smaller in larger crystals. Twisting is one of main residual defects in high-quality crystals and determines the crystal perfection. Furthermore, it is presumed that the handedness of twisting can be ascribed to the anisotropic interaction of chiral protein molecules associated with asymmetric units in the crystal forms. This mechanism of twisting may correspond to the geometric frustration proposed as a primary mechanism of twisting in molecular crystals. Our finding provides insights for the understanding of growth mechanism and the growth control of high-quality crystals.

Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction

Biologically active vitamin B6-derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.

Protein Crystal Growth Simply by Concentration

Protein Crystal Growth Simply by Concentration

Population-balance study of protein crystal growth from solution using a hyperbolic rate law

Kinetic data for crystal size, recently reported for bulk protein crystal growth from solution, is shown to obey a hyperbolic rate law beginning at some critical time after the commencement of the growth run. Given this rate law, the linear growth rate is established and mass-balance methods are then used to derive analytic time dependent expressions for the nucleation rate, the supersaturation and the distribution function for crystal sizes. Our population-balance approach involves a truncated Fokker–Planck equation along with an integro-differential equation for mass conservation. This model is used to describe three cases of protein crystal growth from solution for which kinetic data has been reported: lysozyme, β–lactoglobulin and insulin. Additionally, we show that this empirical rate law for crystal size is approximately related to the Burton-Cabrera-Frank (BCF) and Kossel-Stranski (KS) growth rates. Further, insight into the so called two-step, process is obtained. For two of the growth cases studied here, two-step activity was experimentally confirmed. The hyperbolic tangent rate law most accurately describes growth beginning at some critical time when two-step activity begins to wane and the bulk crystal phase becomes the more dominate form in the solution. Therefore, there seems to be a transition between growth modes at the critical time. We propose here that two-step activity creates a diffusion limited growth situation in the solution for times less than the critical time. The kinetics of crystal size are estimated, during the early diffusion limited time period, using a known model for diffusion limited growth leading to the crystal radius being directly proportional to the square root of time. At the critical time, the two models meet and the kinetics of the crystal radius can thereafter be described using the hyperbolic rate law. Additionally, features of the hyperbolic growth rate model are used to develop a lever rule that can be used to predict the critical time when the transition between growth modes occurs.

Interaction of vapor cloud and its effect on evaporation from microliter coaxial well

The evaporation of volatile heavy hydrocarbon liquid from coaxial well configuration is studied to understand the vapor phase transport, vapor cloud interaction and its effect on the local evaporation rate of two evaporating coaxial cavities of microliter volume. The gap between the two coaxial cavities is varied without varying the dimensions (width and depth) of the coaxial cavities for studying the vapor cloud interaction. Digital holographic Interferometry (DHI) is used to decipher the vapor mole fraction field above the coaxial well. Gravimetric measurement has been carried out for measuring the evaporation rate. Diffusion-limited simulation of evaporation process has been carried out using COMSOL Multiphysics software to understand the role of convective motion. IR thermography measurement of liquid interface temperature has been carried out to correlate the vapor cloud interaction with local evaporation rate and evaporation induced cooling. A flat-disk shaped vapor cloud surrounds the coaxial cavities. Total evaporation rate measured from DHI shows good agreement with the gravimetric measurement having maximum deviation of 3%. Good match between the evaporation rate measured from gravimetry and holography with diffusion limited model is observed at low Grashof number. The mismatch in evaporation rate with the diffusion limited model is observed at high Grashof number due to higher radius of the coaxial cavity indicating the dominance of convective motion. The evaporation rate from individual well decreases when liquid simultaneously evaporates from the inner and outer coaxial cavities. This reduction in evaporation rate increases with decrease in gap between the two coaxial cavities due to increase in interaction between the vapor cloud. The decrease in local evaporation rate from individual cavity due to presence of a neighboring evaporating cavity correlates with the evaporative cooling effect using temperature measurement of the interface from IR thermography. Vapor cloud interactions of microliter volume coaxial cavities can influence the evaporation rate of individual coaxial cavity and the convection inside the liquid phase. The present study demonstrates the capability to precisely control the evaporation process by appropriate design of coaxial well configuration. The results from the present study can be useful for controlling the evaporation rate in several applications i.e. protein crystal growth, DNA/RNA sequencing and microfabrication etc. by suitable design of coaxial well.

Growth of protein crystals in high-strength hydrogels with the dialysis membrane

Growth of protein crystals in high-strength hydrogels with the dialysis membrane

Nucleation and growth of a single suspended protein crystal by merging phase separated droplets

Nucleation and growth of a single suspended protein crystal by merging phase separated droplets

Growing protein crystals under different conditions

Growing protein crystals under different conditions