Heterogeneous nucleation of protein crystals on fluorinated layered silicate

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface.     

Monomer-dependent secondary nucleation in amyloid formation

Secondary nucleation of monomers on the surface of an already existing aggregate that is formed from the same kind of monomers may lead to autocatalytic amplification of a self-assembly process. Such monomer-dependent secondary nucleation occurs during the crystallization of small molecules or proteins and self-assembled materials, as well as in protein self-assembly into fibrous structures. Indications of secondary nucleation may come from analyses of kinetic experiments starting from pure monomers or monomers supplemented with a low concentration of pre-formed aggregates (seeds). More firm evidence requires additional experiments, for example those employing isotope labels to distinguish new aggregates arising from the monomer from those resulting from fragmentation of the seed. In cases of amyloid formation, secondary nucleation leads to the formation of toxic oligomers, and inhibitors of secondary nucleation may serve as starting points for therapeutic developments. Secondary nucleation displays a high degree of structural specificity and may be enhanced by mutations or screening of electrostatic repulsion.     

Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens

Protein crystallization is a major bottleneck in protein X-ray crystallography, the workhorse of most structural proteomics projects. Because the principles that govern protein crystallization are too poorly understood to allow them to be used in a strongly predictive sense, the most common crystallization strategy entails screening a wide variety of solution conditions to identify the small subset that will support crystal nucleation and growth. We tested the hypothesis that more efficient crystallization strategies could be formulated by extracting useful patterns and correlations from the large data sets of crystallization trials created in structural proteomics projects. A database of crystallization conditions was constructed for 755 different proteins purified and crystallized under uniform conditions. Forty-five percent of the proteins formed crystals. Data mining identified the conditions that crystallize the most proteins, revealed that many conditions are highly correlated in their behavior, and showed that the crystallization success rate is markedly dependent on the organism from which proteins derive. Of the proteins that crystallized in a 48-condition experiment, 60% could be crystallized in as few as 6 conditions and 94% in 24 conditions. Consideration of the full range of information coming from crystal screening trials allows one to design screens that are maximally productive while consuming minimal resources, and also suggests further useful conditions for extending existing screens.     

Surface-enhanced nucleation of insulin amyloid fibrillation

Proteins can interact with biological surfaces such as cell membrane, chaperones, cornea, bone, arteries, veins, and heart cavities of the cardiovascular system and also with non-biological surfaces including dialysis membranes and tubing, catheters, invasive surgical instruments, needles, and artificial implants. Fibrillation of amyloid proteins is implicated in many human diseases, including Alzheimer’s, Parkinson’s, and type II diabetes. Here, we show that heterogeneous surfaces accelerate the human insulin nucleation process that is the rate-determining step during amyloid fibril formation. The observed shorter lag (nucleation) phase correlates both with surface wettability and surface roughness. Surfaces promote faster nucleation possibly by increasing the local concentration of protein molecules. A composite parameter combining both surface wettability and roughness suggests that the ideal surface for slower nucleation should be hydrophilic and smooth. These findings provide a basis for designing suitable biomaterials and biomedical devices, especially those to resist amyloidosis.     

The physical significance of Km in the prothrombinase reaction

Key kinetic parameters for the prothrombinase complex formed on membranes of phosphatidylserine (PS)/phosphatidylcholine (PC) (40/60) (Km = 0.12 microM, kcat = 11 s-1) or PS/PC (2/98) (Km = 0.40 microM, kcat = 11 s-1) differed only slightly. In contrast, the density of proteins on the membrane surface at the km differed greatly for the two membranes. The kinetics appeared unaffected by conditions where the number of phospholipid vesicles (2% PS) exceeded the number of protein molecules. These results establish that the Km for the prothrombinase reaction is determined by the concentration of prothrombin in solution rather than its density at the membrane surface. This system can be treated as a dissociable enzyme acting on a soluble substrate.     

Crystallization mechanisms of hemoglobin C in the R state

Crystallization of the mutated hemoglobin, HbC, which occurs inside red blood cells of patients expressing betaC-globin and exhibiting the homozygous CC and the heterozygous SC (in which two mutant beta-globins, S and C, are expressed) diseases, is a convenient model for processes underlying numerous condensation diseases. As a first step, we investigated the molecular-level mechanisms of crystallization of this protein from high-concentration phosphate buffer in its stable carbomonoxy form using high-resolution atomic force microscopy. We found that in conditions of equilibrium with the solution, the crystals' surface reconstructs into four-molecule-wide strands along the crystallographic a (or b) axis. However, the crystals do not grow by the alignment of such preformed strands. We found that the crystals grow by the attachment of single molecules to suitable sites on the surface. These sites are located along the edges of new layers generated by two-dimensional nucleation or by screw dislocations. During growth, the steps propagate with random velocities, with the mean being an increasing function of the crystallization driving force. These results show that the crystallization mechanisms of HbC are similar to those found for other proteins. Therefore, strategies developed to control protein crystallization in vitro may be applicable to pathology-related crystallization systems.     

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.     

An Auto-Catalytic Surface for Conformational Replication of Amyloid Fibrils—Genesis of an Amyloid World?

Amyloid fibrils are composed of self assembled stacked peptide or protein molecules folded and trapped in a stable cross-beta-sheet conformation. The amyloid fibrillation mechanism represents an intriguing self-catalyzed process rendering replication of a molecular conformational memory of interest for prebiotic chemistry. Herein we describe how a solid surface can be rendered auto-catalytic for fibrillation of a protein solution. We have discovered that a hydrophobic silicon or glass surface can be made to continuously fibrillate solutions of insulin monomers under stressed conditions (pH 1.6, 65°C). It was found that the surface acts as a platform for the formation of nascent seeds that induce fibril replication on and at the surface. This autocatalytic effect stems from a layer a few insulin molecules thick representing an oligomeric layer of misfolded, conformationally trapped, insulin molecules that rapidly through epitaxial growth catalyze the rate determining step (nucleation) during fibril replication. This autocatalytic layer is generated by the protein-solid surface interaction and conformational changes of the adsorbed protein during exposure at the air-water interface. The resulting autocatalytic surface thus both initiates local conformational molecular self-replication and acts as a reservoir for fibril seeds budding off into solution spreading fibril replication entities to the surrounding medium. The possibility of catalysis of the conformational replication process by minute amounts of nucleation sites located on a recruiting surface can evade the issue of dramatic concentration dependence of amyloidogenesis.     

In Situ μGISAXS: I. Experimental Setup for Submicron Study of Protein Nucleation and Growth

In this study, we used microbeam grazing-incidence small-angle x-ray scattering (μGISAXS) to investigate in situ protein nucleation and crystal growth assisted by a protein nanotemplate, and introduced certain innovations to improve the method. Our aim was to understand the protein nanotemplate method in detail, as this method has been shown to be capable of accelerating and increasing crystal size and quality, as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing protein crystals at different stages of nucleation and growth. Two model proteins, lysozyme and thaumatin, were used under unique flow conditions to differentially probe protein crystal nucleation and growth.     

Protein Polymerization into Fibrils from the Viewpoint of Nucleation Theory

The assembly of various proteins into fibrillar aggregates is an important phenomenon with wide implications ranging from human disease to nanoscience. Using general kinetic results of nucleation theory, we analyze the polymerization of protein into linear or helical fibrils in the framework of the Oosawa-Kasai (OK) model. We show that while within the original OK model of linear polymerization the process does not involve nucleation, within a modified OK model it is nucleation-mediated. Expressions are derived for the size of the fibril nucleus, the work for fibril formation, the nucleation barrier, the equilibrium and stationary fibril size distributions, and the stationary fibril nucleation rate. Under otherwise equal conditions, this rate decreases considerably when the short (subnucleus) fibrils lose monomers much more frequently than the long (supernucleus) fibrils, a feature that should be born in mind when designing a strategy for stymying or stimulating fibril nucleation. The obtained dependence of the nucleation rate on the concentration of monomeric protein is convenient for experimental verification and for use in rate equations accounting for nucleation-mediated fibril formation. The analysis and the results obtained for linear fibrils are fully applicable to helical fibrils whose formation is describable by a simplified OK model.     

Crystallization of IgG1 by mapping its liquid-liquid phase separation curves

Monoclonal antibody therapeutics is an important and fast expanding market. While production of these molecules has been a major area of research, much less is known regarding the stabilization of these proteins for delivery as drugs. Crystallization of antibodies is one such promising route for protein stabilization at high titers, and here we took a systematic approach to initiate crystallization through nucleation in a simple PEG (polyethylene glycol), protein in water solution. A ternary mixture of globular proteins, PEG, and water will undergo a liquid-liquid phase separation (LLPS) as shown in a phase diagram or a Binodal curve. Of particular interest within the phase diagram is the position of the critical point, which is where nucleation occurs most rapidly. Detailed LLPS maps were created by increasing concentrations of PEG (from 5% to 11%) and IgG (from 1 to 20 mg/mL). By increasing the molecular weight (MW) of PEG (and hence its radius of gyration) from 1,000 to 6,000 g/mol, the temperatures of the critical point of nucleation were shown to increase. Once these curves were determined, nucleation experiments were conducted close to a chosen critical point (10.5 mg/mL IgG in 11% PEG 1000) and after 3 weeks, crystals of IgG of approximately 100 microm in size were successfully formed. This is the first example of crystallization of an antibody through systematic mapping of LLPS curves, which is a fundamental step towards the scale-up of antibody crystallization.     

Kinetic analysis of protein crystal nucleation in gel matrix

The effect of agarose on nucleation of hen egg white lysozyme crystal was examined quantitatively using a temperature-jumping technique. For the first time, to our knowledge, the inhibition of agarose during the nucleation of lysozyme was quantified in two respects: a), the effect of increasing interfacial nucleation barrier, described by the so-called interfacial correlation parameter f(m); and b), the ratio of diffusion to interfacial kinetics obtained from dynamic surface tension measurements. It follows from a dynamic surface tension analysis that the agarose network inhibits the nucleation of lysozyme by means of an enhancement of the repulsion and interfacial structure mismatch between foreign bodies and lysozyme crystals, slowing down the diffusion process of the protein molecules and clusters toward the crystal-fluid interface and inhibiting the rearrangement of protein molecules at the interface. Our results, based on ultraviolet-visible spectroscopy, also show no evidence of the supersaturation enhancement effect in protein agarose gels. The effects of nucleation suppression and transport limitation in gels result in bigger, fewer, and perhaps better quality protein crystals. The understandings obtained in this study will improve our knowledge in controlling the crystallization of proteins and other biomolecules.     

Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles

The conformational changes in well-characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm-whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the "soft" protein molecules (myoglobin, hemoglobin, and BSA), while they were insingnificant in the "rigid" proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in alpha-helix content upon adsorption. Moreover, the native alpha-helix content, which is given as the percentage of the alpha-helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well-known hypotheses for the confirmational chages in protein molecules upon adsorption. (c) 1992 John Wiley & Sons, Inc.     

A novel dialysis procedure for the crystallization of proteins

Various dialysis methods are commonly employed for the crystallization of proteins. Typical procedures include the use of dialysis bags, dialysis buttons or Zeppezauer microdiffusion cells. The general principle involved is that the protein solution is gradually brought to a point of supersaturation by imposing a gradient of ionic strength or organic solvent concentration across the wall of the dialysis membrane. However, in some cases, the imposition of this gradient across the dialysis membrane can result in the formation of a large number of crystal nucleation sites, thereby giving rise to a reduction in the maximum size of the crystals which can be obtained. A novel 'double-dialysis' procedure which incorporates a second dialysis membrane, thus reducing the rate of equilibration in the crystallization experiment, has been developed in our laboratory. The system has been employed successfully on the delta toxin of Staphylococcus aureus resulting in a useful increase in crystal size. A more quantitative analysis of the technique has been carried out on rat liver malic enzyme. The results of a limited series of crystallization trials with this protein have shown that employment of the 'double-dialysis' technique allows a fine control of the rate of crystal nucleation and therefore provides a mechanism for the controlled growth of large crystals.     

The interaction of bovine factor IX, its activation intermediate, factor IX alpha, and its activation products, factor IXa alpha and factor IXa beta, with acidic phospholipid vesicles of various compositions.

The interactions of bovine factor IX, its activation intermediate, Factor IX alpha, and its activation products, Factor IXa alpha and Factor IXa beta, with phospholipid vesicles, of mean radius of approx. 30 nm, containing various amounts of phosphatidylserine (PS) and phosphatidylcholine (PC), have been examined. For Factor IX, Factor IX alpha, Factor IXa alpha and Factor IXa beta, the dissociation constants, at saturating levels of Ca2+, are independent of the PS concentration in the vesicle after levels of 20-30% (w/w) have been reached, and attain minimum values of approx. 1.7, 1.7, 0.7 and 1.0 microM, respectively, with vesicles containing 50% PS. The amount of protein bound per vesicle particle is independent of the PS content, above 20% PS, for Factor IX and Factor IXa beta, with values of approx. 995-1197 and 1128-1566 molecules/vesicle, respectively. With Factor IX alpha, a dependence on the amount of protein bound with the content of PS is seen, which ranges from 338 to 619 molecules/vesicle with membranes containing 30-50% PS. For Factor IXa alpha, no regularity is noted and a range of 583-1083 molecules of protein/vesicle is observed with the systems employed. Examination of the radii of the proteins on the vesicle demonstrates that Factors IX alpha and IXa alpha occupy considerably more of the surface than do Factors IX and IXa beta, suggesting that a reason for the decreased number of binding sites for the former two proteins on the vesicle may be related to their greater surface spatial requirements.     

Zero-sized effect of nano-particles and inverse homogeneous nucleation. Principles of freezing and antifreeze

It was found that freezing of water in terms of homogeneous nucleation of ice never occurs even in ultra-clean micro-sized water droplets under normal conditions. More surprisingly, at sufficiently low supercoolings, foreign nano-particles exert no effect on the nucleation barrier of ice; it is as if they physically "vanished." This effect, called hereafter the "zero-sized" effect of foreign particles (or nucleators), leads to the entry of a so-called inverse homogeneous-like nucleation domain, in which nucleation is effectively suppressed. The freezing temperature of water corresponds to the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation. The freezing temperature of water is mainly determined by (i) the surface roughness of nucleators at large supercoolings, (ii) the interaction and structural match between nucleating ice and the substrate, and (iii) the size of the effective surface of nucleators at low supercoolings. Our experiments showed that the temperature of -40 degrees C, commonly regarded as the temperature of homogeneous nucleation-mediated freezing, is actually the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation in ultra-clean water. Taking advantage of inverse homogeneous-like nucleation, the interfacial tensions between water and ice in very pure water and antifreeze aqueous solutions were measured at a very high precision for the first time. The principles of freezing promotion and antifreeze and the selection for the biological ice nucleation and antifreeze proteins are obtained. The results provide completely new insights into freezing and antifreeze phenomena and bear generic implications for all crystallization systems.     

Thermodynamics of the hydrophobicity in crystallization of insulin

For insight into the solvent structure around protein molecules and its role in phase transformations, we investigate the thermodynamics of crystallization of the rhombohedral form of porcine insulin crystals. We determine the temperature dependence of the solubility at varying concentration of the co-solvent acetone, Cac=0%, 5%, 10%, 15%, and 20%, and find that, as a rule, the solubility of insulin increases as temperature increases. The enthalpy of crystallization, undergoes a stepwise shift from approximately -20 kJ mol(-1) at Cac=0%, 5%, and 10% to approximately -55 kJ mol(-1) at Cac=15% and 20%. The entropy change upon crystallization is approximately 35 J mol(-1) K(-1) for the first three acetone concentrations, and drops to approximately -110 J mol(-1) K(-1) at Cac=15% and 20%. DeltaS degrees cryst>0 indicates release of solvent, mostly water, molecules structured around the hydrophobic patches on the insulin molecules' surface in the solution. As Cac increases to 15% and above, unstructured acetone molecules apparently displace the waters and their contribution to DeltaS degrees cryst is minimal. This shifts DeltaS degrees cryst to a negative value close to the value expected for tying up of one insulin molecule from the solution. The accompanying increase in DeltaH degrees cryst suggests that the water structured around the hydrophobic surface moieties has a minimal enthalpy effect, likely due to the small size of these moieties. These findings provide values of the parameters needed to better control insulin crystallization, elucidate the role of organic additives in the crystallization of proteins, and help us to understand the thermodynamics of the hydrophobicity of protein molecules and other large molecules.     

Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation

Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.     

Surface modification of polystyrene Petri dishes by plasma polymerized 4,7,10-trioxa-1,13-tridecanediamine for enhanced culturing and migration of bovine aortic endothelial cells

Abstract

Plasma surface modification is an effective method for changing material properties to control cell behavior on a surface. This study investigates the efficiency of a plasma polymerized 4,7,10-trioxa-1,13-tridecanediamine (ppTTDDA) film coated on a polystyrene (PS) Petri dish, which is a biocompatible surface with carbon- and oxygen-based chemical species. The adhesion, proliferation, and migration properties of bovine aortic endothelial cells (BAECs) were profoundly enhanced in the ppTTDDA-coated PS Petri dishes without extracellular matrix (ECM) proteins, when compared with the uncoated PS Petri dishes. These observations indicate that ppTTDDA-coated PS Petri dishes can directly interact with cells, regardless of cell adhesion molecules. The increased cell affinity was attributed to the high concentration of carboxyl group on the surface of the ppTTDDA film. Such a carboxyl surface showed an excellent ability to promote culturing of BAECs. Plasma surface modification techniques are effective in improving biocompatibility and provide a surface environment for cell culture.     

Protein purification by bulk crystallization: the recovery of ovalbumin

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems. (c) 1995 John Wiley & Sons, Inc.