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.     

The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.     

Seeded isothermal batch crystallization of lysozyme

The kinetics of lysozyme crystallization under seeded isothermal batch conditions was followed by measurement of the decline in solution concentration versus time. Kinetics were measured for five different values of the seed crystal mass. The data were analyzed using a recently proposed mathematical model. For each seed mass, the model fit the kinetic data well. Growth rate constants determined using the model were approximately constant over a sixfold increase in the seed crystal mass, and fell well within the range of values reported in the literature, but obtained using entirely different experimental techniques. These results confirmed the utility of the proposed model. The proposed model can be used to analyze crystallization kinetics using absorbance measurements only, without the need to characterize the crystal size, thus avoiding the need for expensive laser light scattering and digital microscopy instrumentation. Thus, the model offers a low-cost straightforward method to analyze and simulate the effects of changes in operating parameters such as the seed crystal mass, solution volume, initial protein concentration, pH, temperature, salt concentration, and time.     

Precipitants and additives for membrane crystallization of lysozyme

Membrane crystallization is a newly developed crystallization technique that has proven to be superior in producing good crystal forms under operating conditions that are not appropriate to perform the crystallization process by other traditional techniques. In this work, static membrane crystallization was carried out on lysozyme, with hollow-fiber microporous hydrophobic membranes. Numerous precipitant and additive types and concentrations were employed in the crystallization processes in order to select the most appropriate precipitant and additive types and to find their corresponding concentration levels that can yield the best crystal forms. The crystallization processes were analyzed in two ways: firstly, by evaluation of the transmembrane fluxes obtained by using different precipitants and additives; secondly, by utilization of the images and results obtained from the micrography and IR spectra in comparisons and evaluations of the crystals formed under all kinds of conditions. Moreover, the size distributions of the crystals yielded under several typical crystallization conditions were analyzed, and turbidity and induction time periods obtained during typical crystallization experiments were also measured. Amongst the numerous precipitants and additives tested, the most appropriate precipitant type and additive were chosen and their concentrations were optimized. Good lysozyme crystals were obtained using a certain precipitant and additive. The obtained results from this work further support the advantages of utilizing the membrane crystallization technique for macromolecule crystallizations.     

The mass transfer process and the growth rate of protein crystals

In this paper, protein crystal growth is studied by a Mach-Zehnder interferometer and an image process system. The interference fringe images are recorded during the crystallization of tetragonal hen egg white lysozyme crystal. Concentration distributions of the protein solution are given from the interference fringe images recorded by the Mach-Zehnder interferometer with a real time servo system of a four-step phase shift. The mass transfer flux and the crystal growth rates are obtained from the concentration distribution. The results show that the observed rates are in accordance with those demonstrated by measurements of the experimental images; therefore the method for determining growth rate by the diffusion process is reasonable.     

Differential pathways in oxy and deoxy HbC aggregation/crystallization

CC individuals, homozygous for the expression of beta(C)-globin, and SC individuals expressing both beta(S) and beta(C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype. To date, the question remains as to why HbC forms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal formation induced under conditions of concentrated phosphate buffer (2g% Hb, 1. 8M potassium phosphate buffer) and viewed by differential interference contrast microscopy. Oxy HbC formed isotropic amorphous aggregates with subsequent tetragonal crystal formation. Also observed, but less numerous, were twisted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in the oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored the formation of a greater morphologic variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed flat, hexagonal crystals. These results suggest: (1) the Lys substitution at beta6 evokes a crystallization process dependent upon ligand state conformation [i. e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) the oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of HbC that energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.     

Protein crystallization by design: chymotrypsinogen without precipitants

Protein crystals are usually obtained by an empirical approach based on extensive screening to identify suitable crystallization conditions. In contrast, we have used a systematic predictive procedure to produce data-quality crystals of bovine chymotrypsinogen A and used them to obtain a refined X-ray structure to 3 A resolution. Measurements of the osmotic second virial coefficient of chymotrypsinogen solutions were used to identify suitable solvent conditions, following which crystals were grown for approximately 30 hours by ultracentrifugal crystallization, without the use of any precipitants. Existing structures of chymotrypsinogen were obtained in solutions including 10-30 % ethanol, whereas simple buffered NaCl solutions were used here. The protein crystallized in the tetragonal space group P4(1)2(1)2, with one molecule per asymmetric unit. The quality of the refined map was very high throughout, with the main-chain atoms of all but four residues clearly defined and with nearly all side-chains also defined. Although only minor differences are seen compared to the structures previously reported, they indicate the possibility of structural changes due to the crystallization conditions used in those studies. Our results show that more systematic crystallization of proteins is possible, and that the procedure can expand the range of conditions under which crystals can be grown successfully and can make new crystal forms available.     

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

An X-ray study of the physiological-temperature form of hen egg-white lysozyme at 2 A resolution

The structure of the high-temperature orthorhombic form of hen egg-white lysozyme has been determined at 2.0 A resolution. Initial images of the molecule were obtained at 6.0 A resolution both by double isomorphous replacement and by molecular replacement with use of the known structure of the room-temperature tetragonal lysozyme. The initial model thus obtained (R = 0.52 at 6.0 A) was refined first as a rigid body at 6.0 A and then by restrained least squares at 2.5 A and later at 2.0 A resolution. The final model (R = 0.23 at 2.0 A) was compared with that of the tetragonal form: the structures are very similar with a root mean square difference in superimposed alpha-carbon coordinates of 0.46 A. There are, however, differences which are caused by a crystal contact involving the upper part of this active site in the high-temperature orthorhombic form. Because of this, residues Trp 62 and Pro 70 are much better ordered than in the tetragonal form, where they are exposed to solvent. These differences can partly explain the difficulty of inhibitor-binding in high-temperature orthorhombic crystals, but do not seem to reflect the particular behaviour of lysozyme in solution at high temperature.     

Enhancing protein crystallization through precipitant synergy

Suitable conditions for protein crystallization are commonly identified by screening combinations of independent factors that affect crystal formation. Because precipitating agents are prime determinants of crystallization, we investigated whether a systematic exploration of combinations of mechanistically distinct precipitants would enhance crystallization. A crystallization screen containing 64 precipitant mixtures was devised. Tests with ten HIV envelope-related proteins demonstrated that use of precipitant mixtures significantly enhanced both the probability of crystallization as well as the quality of optimized crystals. Tests with hen egg white lysozyme generated a novel C2 crystal from a salt/organic solvent mixture; structure solution at 2 A resolution revealed a lattice held together by both hydrophobic and electrostatic dyad interactions. The results indicate that mechanistically distinct precipitants can synergize, with precipitant combinations adding unique dimensions to protein crystallization.     

Kinetic resolution of peptide bond and side chain far-UV circular dichroism during the folding of hen egg white lysozyme.

The kinetics of regain of the native ellipticity in the far- and near-UV spectra have been investigated during the refolding at pH 7.8 and 20 degrees C of guanidine-unfolded, nonreduced hen egg white lysozyme. Stopped-flow studies showed that the ellipticities at 260 and 289.5 nm exhibit biphasic kinetics with rate constants of about 50 s-1 and 2.5 s-1 for the rapid and slow phase, respectively. The ellipticity in the far-UV obeyed triphasic kinetics. In addition to a rapid and a slow phase with rate constants similar to those observed in the near-UV, a "burst" of ellipticity was shown to occur in the dead time of the experiments. The effects of low pH and of concentrations of guanidine ranging from 0.075 to 1.5 M on the rapid and slow rate constants were studied. Under all conditions investigated, the rate constants observed in the far- and near-UV for a given phase were the same, thus suggesting that the molecular events observed in the two regions of the UV spectrum are either identical or strongly coupled. Continuous-flow experiments at different wavelengths between 214 and 240 nm under conditions where the dead time for the observation was only 4 ms, followed by a detailed analysis of the kinetics of ellipticity change at each wavelength, provided the spectrum of the molecular species formed at the end of the burst phase. This spectrum was found to closely fit that predicted from the secondary structure of native lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and equilibria of lysozyme precipitation and crystallization in concentrated ammonium sulfate solutions

The kinetics and thermodynamics of lysozyme precipitation in ammonium sulfate solutions at pH 4 and 8 and room temperature were studied. X-ray powder diffraction (XRD) was used to characterize the structure of lysozyme precipitates. It was found that, if sufficient time was allowed, microcrystals developed following an induction period after initial lysozyme precipitation, even up to ionic strengths of 8 m and at acidic pH, where lysozyme is refractory to crystallization in ammonium sulfate. The full set of precipitation and crystallization data allowed construction of a phase diagram of lysozyme, showing the ammonium sulfate dependence. It suggests that precipitation may reflect a frustrated metastable liquid-liquid phase separation, which would allow this process to be understood within the framework of the generic phase diagram for proteins. The results also demonstrate that XRD, more frequently used for characterizing inorganic and organic polycrystalline materials, is useful both in characterizing the presence of crystals in the dense phase and in verifying the crystal form of proteins.     

Refined structures of Bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment

The HyHEL-5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg-white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL-5, an anti-hen egg-white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side-chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL-5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side-chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL-5 is similar to that in previously determined lysozyme-HyHEL-5 complexes. © 1996 Wiley-Liss, Inc.     

A nuclear magnetic resonance study of the hydrogen-exchange behaviour of lysozyme in crystals and solution

Amide hydrogen/deuterium exchange behaviour has been studied for all of the peptide amides of hen lysozyme by means of two-dimensional n.m.r. spectroscopy. The amides have been grouped into four categories on the basis of their rates of exchange in solution at pH 4.2 and 7.5. The distribution of the amides into the different categories has been examined in the light of the crystallographic structural information, considering the type of secondary structure, the nature of hydrogen bonding and the distance from the protein surface. None of these features was found to determine uniquely the pattern of hydrogen exchange rates within the protein. The exchange behaviour of the individual amides could, however, in general be rationalized by a combination of these features. Hydrogen exchange was also monitored in both tetragonal and triclinic crystals of lysozyme, by allowing exchange to take place in the crystals prior to dissolution and recording of n.m.r. spectra under conditions where further exchange was minimized. This enabled direct comparison to be made of the exchange behaviour in the crystals and solution. A reduction in exchange rate was observed in the crystalline state relative to solution for a substantial number of amides and distinct differences between exchange in the different crystals could be observed. These differences between the solution and the different crystal states do not, however, correlate in a simple manner with proximity to intermolecular contacts in the crystals. However, the existence of these contacts, which are on the surface of the protein molecule, have a profound effect on the exchange of amides in the interior of the protein. The results indicate that the spectrum of fluctuations giving rise to hydrogen exchange may be significantly altered by the intermolecular interactions present within the crystalline state.     

Rotation function studies of human C-reactive protein

Rotation function studies of two tetragonal crystal forms of human C-reactive protein have confirmed the pentameric structure of the molecule. The two crystal forms have space groups P4122 (I) and P4222 (II) with closely similar unit cells and are often twinned together. Investigation of the crystallization conditions indicates that dissociation heterogeneity has been a major limiting factor in the reproducible growth of good single crystals. The orientation of the pentameric molecule is shown to be almost identical in both forms, about the axial direction omega = 57 degrees, phi = 45 degrees, i.e. 57 degrees away from c in the (110) plane.     

Structural study on hen egg-white lysozyme crystals grown in gravity and microgravity

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离子液体1-丁基-3-甲基咪唑氯代盐对溶菌酶结晶的影响

以亲水性离子液体1-丁基-3-甲基咪唑氯盐(BmimCl)为添加剂,研究离子液体对溶菌酶结晶的影响.分别考察了离子液体对溶菌酶晶体数量与尺寸、晶体形貌及蛋白质纯度的影响,并探讨了离子液体对结晶过程影响的作用机制.离子液体通过增大溶菌酶的溶解度和其自身低蒸气压两种途径,降低了溶菌酶在结晶过程中的过饱和度,更有利于晶体的成核和生长,得到更好的结果.如避免多晶态现象的发生,增大晶体的尺寸,降低溶菌酶样品纯度的要求.X-射线衍射分析表明,离子液体未改变晶体的晶型结构,但可提高晶体的衍射分辨率.     

Protein crystal growth. Growth kinetics for tetragonal lysozyme crystals

A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where R = rate in cm/s; kappa = constant; sigma i = solute growth interface supersaturation; and r = rate dependence upon super-saturation, with the result that kappa = 0.146 X 10(-8) cm/s and r = 2.0. A model of the growth process was developed and the experimental data were used to determine the relative roles of transport and interfacial kinetics in the growth of this crystal. Values for the width of the boundary layer delta, the interfacial concentration Ci, and growth rate R were determined. The model may be used to extrapolate to other growth conditions. The relative role of transport and interfacial kinetics can be expressed by the coefficient gamma = (CB - Ci)/(CB - Cs), when CB is the bulk concentration and Cs the saturation. Values for gamma were found to range from much less than 0.1 for submicron-size crystals to approximately 0.15 for cm sizes. The results indicate that attachment or surface effects are rate-limiting in lysozyme crystal growth in Earth's gravity because solutal convection always provides more transport of solute than can be accommodated by the interface. In order to grow such crystals under transport limiting conditions, it would be necessary to suppress this solutal convection.     

Structure of a second crystal form of Bence-Jones protein Loc: strikingly different domain associations in two crystal forms of a single protein

We have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-A resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding "pocket". The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion (Chang et al., 1985). The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry.     

Crystal structure of Tapes japonica Lysozyme with substrate analogue: structural basis of the catalytic mechanism and manifestation of its chitinase activity accompanied by quaternary structural change

Tapes japonica lysozyme (TJL) is classified as a member of the recently established i-type lysozyme family. In this study, we solved the crystal structure of TJL complexed with a trimer of N-acetylglucosamine to 1.6A resolution. Based on structure and mutation analyses, we demonstrated that Glu-18 and Asp-30 are the catalytic residues of TJL. Furthermore, the present findings suggest that the catalytic mechanism of TJL is a retaining mechanism that proceeds through a covalent sugar-enzyme intermediate. On the other hand, the quaternary structure in the crystal revealed a dimer formed by the electrostatic interactions of catalytic residues (Glu-18 and Asp-30) in one molecule with the positive residues at the C terminus in helix 6 of the other molecule. Gel chromatography analysis revealed that the TJL dimer remained intact under low salt conditions but that it dissociated to TJL monomers under high salt conditions. With increasing salt concentrations, the chitinase activity of TJL dramatically increased. Therefore, this study provides novel evidence that the lysozyme activity of TJL is modulated by its quaternary structure.