Cloud-point temperature and liquid-liquid phase separation of supersaturated lysozyme solution

The detailed understanding of the structure of biological macromolecules reveals their functions, and is thus important in the design of new medicines and for engineering molecules with improved properties for industrial applications. Although techniques used for protein crystallization have been progressing greatly, protein crystallization may still be considered an art rather than a science, and successful crystallization remains largely empirical and operator-dependent. In this work, a microcalorimetric technique has been utilized to investigate liquid-liquid phase separation through measuring cloud-point temperature T(cloud) for supersaturated lysozyme solution. The effects of ionic strength and glycerol on the cloud-point temperature are studied in detail. Over the entire range of salt concentrations studied, the cloud-point temperature increases monotonically with the concentration of sodium chloride. When glycerol is added as additive, the solubility of lysozyme is increased, whereas the cloud-point temperature is decreased.     

Attempts to rationalize protein crystallization using relative crystallizability

Protein crystal growth (PCG) remains the bottleneck of crystallography despite many decades of study. The nucleation zone in the two-dimensional-phase diagram has been used to evaluate the relative crystallizability of proteins, which is expressed as a percentage over the phase area delineated by experimental protein and precipitating agent concentration ranges. For protein-salts which are subject to a direct temperature effect on solubility, as represented by Egg Lysozyme, a decrease in temperature augments the nucleation zone percentage whereas for those with retrograde solubility as a function of temperature, for example fructose-1,6-bisphosphatase in the presence and absence of AMP, an increase in temperature can significantly enhance the relative crystallizability. These results have been confirmed by the number of "hits" using PEGs as precipitating agents in Sparse Matrix Screen experiments for different proteins and are in excellent agreement with the relative crystallizability. The relationship between solubility dependence, relative crystallizability and crystallization success, has been evidenced. Such crystallizability can become a guide to identify efficient crystallization regions, providing a rational approach to PCG and structural biology.     

The effect of ionic strength, temperature, and pressure on the interaction potential of dense protein solutions: from nonlinear pressure response to protein crystallization

Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2 values for controlled protein crystallization.     

Molecular origins of osmotic second virial coefficients of proteins.

The thermodynamic properties of protein solutions are determined by the molecular interactions involving both solvent and solute molecules. A quantitative understanding of the relationship would facilitate more systematic procedures for manipulating the properties in a process environment. In this work the molecular basis for the osmotic second virial coefficient, B22, is studied; osmotic effects are critical in membrane transport, and the value of B22 has also been shown to correlate with protein crystallization behavior. The calculations here account for steric, electrostatic, and short-range interactions, with the structural and functional anisotropy of the protein molecules explicitly accounted for. The orientational dependence of the protein interactions is seen to have a pronounced effect on the calculations; in particular, the relatively few protein-protein configurations in which the apposing surfaces display geometric complementarity contribute disproportionately strongly to B22. The importance of electrostatic interactions is also amplified in these high-complementarity configurations. The significance of molecular recognition in determining B22 can explain the correlation with crystallization behavior, and it suggests that alteration of local molecular geometry can help in manipulating protein solution behavior. The results also have implications for the role of protein interactions in biological self-organization.     

Static light scattering studies of OmpF porin: implications for integral membrane protein crystallization

Integral membrane proteins carry out some of the most important functions of living cells, yet relatively few details are known about their structures. This is due, in large part, to the difficulties associated with preparing membrane protein crystals suitable for X-ray diffraction analysis. Mechanistic studies of membrane protein crystallization may provide insights that will aid in determining future membrane protein structures. Accordingly, the solution behavior of the bacterial outer membrane protein OmpF porin was studied by static light scattering under conditions favorable for crystal growth. The second osmotic virial coefficient (B22) was found to be a predictor of the crystallization behavior of porin, as has previously been found for soluble proteins. Both tetragonal and trigonal porin crystals were found to form only within a narrow window of B22 values located at approximately -0.5 to -2 X 10(-4) mol mL g(-2), which is similar to the "crystallization slot" observed for soluble proteins. The B22 behavior of protein-free detergent micelles proved very similar to that of porin-detergent complexes, suggesting that the detergent's contribution dominates the behavior of protein-detergent complexes under crystallizing conditions. This observation implies that, for any given detergent, it may be possible to construct membrane protein crystallization screens of general utility by manipulating the solution properties so as to drive detergent B22 values into the crystallization slot. Such screens would limit the screening effort to the detergent systems most likely to yield crystals, thereby minimizing protein requirements and improving productivity.     

Effects of additives on surfactant phase behavior relevant to bacteriorhodopsin crystallization

The interactions leading to crystallization of the integral membrane protein bacteriorhodopsin solubilized in n-octyl-beta-D-glucoside were investigated. Osmotic second virial coefficients (B(22)) were measured by self-interaction chromatography using a wide range of additives and precipitants, including polyethylene glycol (PEG) and heptane-1,2,3-triol (HT). In all cases, attractive protein-detergent complex (PDC) interactions were observed near the surfactant cloud point temperature, and there is a correlation between the surfactant cloud point temperatures and PDC B(22) values. Light scattering, isothermal titration calorimetry, and tensiometry reveal that although the underlying reasons for the patterns of interaction may be different for various combinations of precipitants and additives, surfactant phase behavior plays an important role in promoting crystallization. In most cases, solution conditions that led to crystallization fell within a similar range of slightly negative B(22) values, suggesting that weakly attractive interactions are important as they are for soluble proteins. However, the sensitivity of the cloud point temperatures and resultant coexistence curves varied significantly as a function of precipitant type, which suggests that different types of forces are involved in driving phase separation depending on the precipitant used.     

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.     

Protein solubility modeling.

A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process.     

Correlation of second virial coefficient with solubility for proteins in salt solutions

In this work, osmotic second virial coefficients (B(22)) were determined and correlated with the measured solubilities for the proteins, α-amylase, ovalbumin, and lysozyme. The B(22) values and solubilities were determined in similar solution conditions using two salts, sodium chloride and ammonium sulfate in an acidic pH range. An overall decrease in the solubility of the proteins (salting out) was observed at high concentrations of ammonium sulfate and sodium chloride solutions. However, for α-amylase, salting-in behavior was also observed in low concentration sodium chloride solutions. In ammonium sulfate solutions, the B(22) are small and close to zero below 2.4 M. As the ammonium sulfate concentrations were further increased, B(22) values decreased for all systems studied. The effect of sodium chloride on B(22) varies with concentration, solution pH, and the type of protein studied. Theoretical models show a reasonable fit to the experimental derived data of B(22) and solubility. B(22) is also directly proportional to the logarithm of the solubility values for individual proteins in salt solutions, so the log-linear empirical models developed in this work can also be used to rapidly predict solubility and B(22) values for given protein-salt systems.     

温度影响蛋白质结晶的研究现状

温度控制作为调控蛋白质结晶过程的手段,在结晶实验中被广泛采用。热历史效应作为蛋白质结晶实验中新的影响因素,已被越来越多的科学家所重视。控制温度可以改变蛋白质的溶解度,进一步改变溶液的过饱和度,从而影响结晶过程。我们简要总结了温度对蛋白质结晶的影响及应用温度技术控制蛋白质晶体生长的各种技术,为蛋白质结晶工作提供理论和实验依据。     

Crystal structure of a supercharged variant of the human enteropeptidase light chain

The highly specific serine protease human enteropeptidase light chain cleaves the Asp4Lys recognition sequence and represents an interesting enzyme for biotechnological applications. The human enzyme shows 10 times faster kinetics compared to other animal sources but low solubility under low salt conditions, which hampers protein production and crystallization. Therefore, a supercharged variant (N6D/G21D/G22D/N142D/K210E/C112S) with increased solubility was used for crystallization. The structure (resolution, 1.9 Å) displays a typical α/β trypsin‐like serine protease‐fold. The mutations introduced for protein supercharging generate larger clusters of negative potential on both sites of the active cleft but do not affect the structural integrity of the protein. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Relationship of critical temperature to macromolecular synthesis and growth yield in Psychrobacter cryopegella

Most microorganisms isolated from low-temperature environments (below 4 degrees C) are eury-, not steno-, psychrophiles. While psychrophiles maximize or maintain growth yield at low temperatures to compensate for low growth rate, the mechanisms involved remain unknown, as does the strategy used by eurypsychrophiles to survive wide ranges of temperatures that include subzero temperatures. Our studies involve the eurypsychrophilic bacterium Psychrobacter cryopegella, which was isolated from a briny water lens within Siberian permafrost, where the temperature is -12 degrees C. P. cryopegella is capable of reproducing from -10 to 28 degrees C, with its maximum growth rate at 22 degrees C. We examined the temperature dependence of growth rate, growth yield, and macromolecular (DNA, RNA, and protein) synthesis rates for P. cryopegella. Below 22 degrees C, the growth of P. cryopegella was separated into two domains at the critical temperature (T(critical) = 4 degrees C). RNA, protein, and DNA synthesis rates decreased exponentially with decreasing temperatures. Only the temperature dependence of the DNA synthesis rate changed at T(critical). When normalized to growth rate, RNA and protein synthesis reached a minimum at T(critical), while DNA synthesis remained constant over the entire temperature range. Growth yield peaked at about T(critical) and declined rapidly as temperature decreased further. Similar to some stenopsychrophiles, P. cryopegella maximized growth yield at low temperatures and did so by streamlining growth processes at T(critical). Identifying the specific processes which result in T(critical) will be vital to understanding both low-temperature growth and growth over a wide range of temperatures.     

Analysis and statistics of crystallisation success increase by composition modification of protein and precipitant mixing ratio

The nucleation zone has to be reached for any crystal to grow, and the search for crystallization conditions of new proteins is a trial and error process. Here a convenient screening strategy is studied in detail that varies the volume ratio of protein sample to the reservoir solution in the drop to initiate crystallization that is named "composition modification". It is applied after the first screen and has been studied with twelve proteins. Statistical analysis shows a significant improvement in screening using this strategy. The average improvement of "hits" at different temperatures is between 32 and 42%, for examples, 41.8% ± 14.0% and 35.7% ± 12.4% (± standard deviation) at 288 K and 300 K, respectively. Remarkably, some new crystals were found by composition modification which increased the probability of reaching the nucleation zone to initiate crystallization. This was confirmed by a phase diagram study. It is also demonstrated that composition modification can further increase crystallisation success significantly (1.3 times) after the improvement of "hits" by temperature screening. The trajectories of different composition modifications during vapour diffusion were plotted, further demonstrating that protein crystallizability can be increased by hitting more parts of the nucleation zone. It was also found to facilitate the finding of initial crystals for proteins of low solubility. These proteins gradually become more concentrated during the vapour diffusion process starting from a larger protein solution ratio in the initial mixture.     

Crystallization of bacteriorhodopsin from bicelle formulations at room temperature

We showed previously that high-quality crystals of bacteriorhodopsin (bR) from Halobacterium salinarum can be obtained from bicelle-forming DMPC/CHAPSO mixtures at 37 degrees C. As many membrane proteins are not sufficiently stable for crystallization at this high temperature, we tested whether the bicelle method could be applied at a lower temperature. Here we show that bR can be crystallized at room temperature using two different bicelle-forming compositions: DMPC/CHAPSO and DTPC/CHAPSO. The DTPC/CHAPSO crystals grown at room temperature are essentially identical to the previous, twinned crystals: space group P21 with unit cell dimensions of a = 44.7 A, b = 108.7 A, c = 55.8 A, beta = 113.6 degrees . The room-temperature DMPC/CHAPSO crystals are untwinned, however, and belong to space group C222(1) with the following unit cell dimensions: a = 44.7 A, b = 102.5 A, c = 128.2 A. The bR protein packs into almost identical layers in the two crystal forms, but the layers stack differently. The new untwinned crystal form yielded clear density for a previously unresolved CHAPSO molecule inserted between protein subunits within the layers. The ability to grow crystals at room temperature significantly expands the applicability of bicelle crystallization.     

Anomalous temperature dependence of the thermal expansion of proteins

The temperature dependence of the apparent expansibility of lysozyme and ovalbumin in solution has been measured as a function of pH. This temperature dependence is explained in terms of suppressed fluctuations in bound water due to the protein. It is shown that the thermal expansion coefficient of bound water is different from bulk water. The pH dependence can be explained by increased hydration of side chains at lower pH. The amount in volume of hydration water in a typical protein-water system varies from 0.16 to 0.7. How the intrinsic thermal expansion coefficient of proteins can be derived from the apparent quantity is discussed. Intrinsic values of the thermal expansion coefficient for lysozyme at room temperature are between 1.7 and 4.4 x 10(-4) K-1 for a 10% solution.     

Effect of cosolvents on solubility of protein in equilibrium between different states

This paper describes a theoretical work on the solubility of a protein in equilibrium between different forms such as isomerization (A ? B) and dimerization (2A ? B). Under the assumptions that A and B have different solubilities and form their own solid phases (in the form of either precipitation or crystal), it was shownthat only either A or B precipitates at a given condition, another form being in equilibrium in solution with the precipitating form, provided that the solubility ratio of A to B is not identical to their equilibrium composition in the solution phase determined by the equilibrium constant. It follows, then, that the total concentration of the protein in the solution phase can be calculated by summing the solubility of the precipitating form and the concentration in solution of another form in equilibrium with the precipitating form. Assuming various preferential protein-solvent interactions for the two forms in solution as well as solid phases, dependences of the total protein solubility on the additive concentration were examined. It was shown that this dependence may be complex, showing maximum, minimum, or inflection point, depending on the preferential protein interactions with solvent components.     

Solubility, metastable zone width, and racemic characterization of propranolol hydrochloride

Characterization of the racemic species, which can be a racemic compound, a racemic conglomerate, or a pseudoracemate (solid solution), is a prerequisite for the design of crystallization resolution processes. It is useful to determine the solid/liquid equilibrium solubility of the enantiomer mixtures for crystallization operation. For the beta-blocker drug propranolol hydrochloride, Gibbs free energy of formation of racemic compound and entropy of mixing of the (R)- and (S)- enantiomers in the liquid state for racemic conglomerate were calculated. The structural differences between (R, S)-propranolol hydrochloride and its (S)-enantiomer were further investigated by powder X-ray diffraction patterns, infrared spectra, and solid-state NMR spectra. The solubility and metastable zone width of (R, S)- propranolol hydrochloride in a mixed solvent of methanol and acetone were determined by cooling crystallization over the temperature range 3.5-42.5 degrees C. The ternary solubility diagram of (R)-, (S)-propranolol hydrochloride was constructed using the same mixed solvent. The diagram will be useful as a guide for choosing crystallization operation conditions to produce pure enantiomers.     

Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin

The P23T mutation in the human gammaD-crystallin gene has in recent years been associated with a number of well known cataract phenotypes. To understand the molecular mechanism of lens opacity caused by this mutation, we expressed human gammaD-crystallin (HGD), the P23T mutant, and other related mutant proteins in Escherichia coli and compared the structures and thermodynamic properties of these proteins in vitro. The results show that the cataract-causing mutation P23T does not exhibit any significant structural change relative to the native protein. However, in marked contrast to the native protein, the mutant shows a dramatically lowered solubility. The reduced solubility results from the association of the P23T mutant to form a new condensed phase that contains clusters of the mutant protein. The monomer-cluster equilibrium is represented by a solubility curve in the phase diagram. When the solubility limit is exceeded, the mutant protein forms the condensed phase after a nucleation time of 10-20 min. We found that the solubility of the P23T mutant exhibits an inverse dependence on temperature, i.e., the protein clusters are increasingly soluble as the temperature of the solution decreases. The solubility of P23T can be substantially altered by the introduction of specific mutations at or in the immediate vicinity of residue 23. We examined the mutants P23S, P23V, P23TInsP24, and P23TN24K and found that the latter two mutations can restore the solubility of the P23T mutant. These findings may help develop a strategy for the rational design of small molecule inhibitors of this type of condensed phase.     

Protein crystallization under high pressure

Pressure is expected to be an important parameter to control protein crystallization, since hydrostatic pressure affects the whole system uniformly and can be changed very rapidly. So far, a lot of studies on protein crystallization have been done. Solubility of protein depends on pressure. For instance, the solubility of tetragonal lysozyme crystal increased with increasing pressure, while that of orthorhombic crystal decreased. The solubility of subtilisin increased with increasing pressure. Crystal growth rates of protein also depend on pressure. The growth rate of glucose isomerase was significantly enhanced with increasing pressure. The growth rate of tetragonal lysozyme crystal and subtilisin decreased with increasing pressure. To study the effects of pressure on the crystallization more precisely and systematically, hen egg white lysozyme is the most suitable protein at this stage, since a lot of data can be used. We focused on growth kinetics under high pressure, since extensive studies on growth kinetics have already been done at atmospheric pressure, and almost all of them have explained the growth mechanisms well. The growth rates of tetragonal lysozyme decreased with pressure under the same supersaturation. This means that the surface growth kinetics significantly depends on pressure. By analyzing the dependence of supersaturation on growth rate, it was found that the increase in average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in growth rate. At this stage, it is not clear whether the increase in surface energy with increasing pressure is the main reason or not. Fundamental studies on protein crystallization under high pressure will be useful for high pressure crystallography and high pressure protein science.     

Molecular modeling of temperature dependence of solubility parameters for amorphous polymers

A molecular modeling strategy is proposed to describe the temperature (T) dependence of solubility parameter (δ) for the amorphous polymers which exhibit glass-rubber transition behavior. The commercial forcefield "COMPASS" is used to support the atomistic simulations of the polymer. The temperature dependence behavior of δ for the polymer is modeled by running molecular dynamics (MD) simulation at temperatures ranging from 250 up to 650 K. Comparing the MD predicted δ value at 298 K and the glass transition temperature (T(g)) of the polymer determined from δ-T curve with the experimental value confirm the accuracy of our method. The MD modeled relationship between δ and T agrees well with the previous theoretical works. We also observe the specific volume (v), cohesive energy (U(coh)), cohesive energy density (E(CED)) and δ shows a similar temperature dependence characteristics and a drastic change around the T(g). Meanwhile, the applications of δ and its temperature dependence property are addressed and discussed.