Tetragonal Lysozyme Crystals Research Articles

Effects of a Forced Solution Flow on the Step Advancement on {110} Faces of Tetragonal Lysozyme Crystals: Direct Visualization of Individual Steps under a Forced Solution Flow

To grow protein crystals of better quality, there still exists an open question whether a solution flow should be suppressed or intentionally introduced. To obtain a comprehensive understanding of the effects of a solution flow, we directly measured the velocities of individual elementary steps (on {110} faces of tetragonal lysozyme crystals) under a forced solution flow, for the first time, by laser confocal microscopy combined with differential interference contrast microscopy. When we used crystals grown by a two-dimensional (2D) nucleation growth mechanism in a solution of commercial lysozyme (98.5% purity, from Seikagaku Co.), while increasing the solution flow rate, the step velocity decreased monotonically. We confirmed that this decrease in the step velocity with flow rate was due to the enhancement of the mass transfer of impurity (mainly covalently bonded dimer), by the observation using a lysozyme further purified (dimer was removed). In contrast, when we used crystals grown by a spiral growth mechanism in a commercial lysozyme solution, with increasing the flow rate, the step velocity increased and had the maximum at the flow rate of 10 μm/s, and then decreased monotonically. Also, the step velocity was 2–4 times higher than in the case of the 2D nucleation growth. These results demonstrate that the growth of spiral steps is less affected by impurities because the density of spiral steps is much higher than that of 2D island ones.

Growth Rates of Protein Crystals

Protein crystallization is important for structural biology. The rate at which a protein crystallizes is often the bottleneck in determining the protein's structure. Here, we give a physical model for the growth rates of protein crystals. Most materials crystallize faster under stronger growth conditions; however, protein crystallization slows down under the strongest conditions. Proteins require a crystallization slot of 'just right' conditions. Our model provides an explanation. Unlike simpler materials, proteins are orientationally asymmetrical. Under strong conditions, protein molecules attempt to crystallize too quickly, in wrong orientations, blocking surface sites for more productive crystal growth. The model explains the observation that increasing the net charge on a protein increases the crystal growth rate. The model predictions are in good agreement with experiments on the growth rates of tetragonal lysozyme crystals as a function of pH, salt concentration, temperature, and protein concentration.

Effects of high pressure on the step velocity on the {110} faces of tetragonal lysozyme crystals

We measured step velocity Vstep in the <110> direction of two dimensional islands on the {110} faces of tetragonal lysozyme crystals under high pressure, and calculated step kinetic coefficients βstep. Vstep and βstep decreased with increasing pressure. The decrease in βstep indicates the increase in activation energy of the crystallization εkink or the mean distance between kinks on the step λ.

Solubility measurements by in situ observation of the apex region formed by the (110), (11̄0) and (101) faces of tetragonal lysozyme crystals

Equilibrium temperatures, Te, of tetragonal hen egg-white lysozyme crystals were determined by in situ observation of apex regions formed by the (110), (11̄0) and (101) faces of the crystals. We could precisely measure Te within 60min, and with an error margin ±0.7°C or less. This is largely because growth and dissolution can be detected more rapidly at the apex than at the steps at the center of the crystal faces.

Self-assembly of gold nanoparticles in protein crystal

We developed a method to assemble gold nanoparticles three-dimensionally into a lysozyme crystal. The crystals were formed by incubating a crystallization solution containing lysozyme and gold nanoparticles at 20 °C. Observation of the crystals by an optical microscope revealed that the gold nanoparticles were assembled in {1 0 1} sectors of the tetragonal lysozyme crystal. The distribution coefficients of the gold nanoparticle into the crystal were measured as functions of pH and concentrations of NaCl. It was revealed that the gold nanoparticles are assembled into the crystal by an electrostatic interaction between the gold nanoparticles and the lysozyme on the crystal surface.

Applying broadband terahertz time-domain spectroscopy to the analysis of crystalline proteins: a dehydration study

The application of terahertz time-domain spectroscopy and imaging to the study of proteins in crystalline form is demonstrated. Terahertz time-domain spectroscopy is particularly sensitive to the long-range ordering of molecules, with proven utility for the spectroscopy of crystalline biological small molecules. Here, the terahertz time-domain absorption response of a macromolecular protein single crystal is investigated for the first time. In particular, the effect of dehydration on the terahertz absorption coefficient of tetragonal hen egg white lysozyme crystals is reported.

Effect of various precipitants on the nucleation rate of tetragonal hen egg-white lysozyme crystals in an AC external electric field

It was observed that the effect of an external electric field on the nucleation rate of tetragonal hen egg-white lysozyme crystals varied depending on the precipitant used (NaCl, NiCl 2 or YbCl 3) and that the electric double layer (EDL) played an important role in generating an external electric field of the necessary strength to control the nucleation rate. This phenomenon depended on the ionic strength of the precipitant used; that is, a precipitant of greater ionic strength resulted in a thinner EDL and increased the effect of the external electric field as the driving force for nucleation. The dependence of the nucleation rate on the precipitant was attributed to the magnitudes of the external electric fields generated in EDLs of varying thickness which were formed in the presence of different precipitants.

Estimated effects of silicone glue on protein crystal growth

Silicone glue (modified silicone polymer) is widely used for both experiments involving inorganic crystal growth and those involving organic materials like proteins. This material is very useful for building a hand-made experiment setup or for fixing protein crystals to specific locations. Though silicone glue is regarded as harmful to proteins, no systematic verification was performed to investigate its impurity effects on protein crystal growth. We focused on and estimated the impurity effects of silicone glue on protein crystal growth. Hen egg white lysozyme (HEWL) was used as a model protein. Surface morphology and step velocity of tetragonal lysozyme crystals in the presence and absence of silicone glue were investigated by laser confocal interference contrast microscopy (LCM-DIM). The surface morphology of a tetragonal lysozyme crystal in the presence of silicone glue corresponded to that grown in a lysozyme solution without silicone glue. The dependency of step velocities on supersaturation in the presence of silicone glue also exhibited the same tendency as that of a glue-free system. These two phenomena indicate that the silicone glue did not act as an impurity on lysozyme crystals. Therefore, we conclude that silicone glue is an effective material for various unique experiments involving protein crystals or for applying new methods to create large, high-quality protein crystals.

Growth Rate Dispersion in Protein Crystal Growth

Like many small molecule materials, tetragonal lysozyme crystals exhibit growth rate dispersion. To investigate this phenomenon further, the growth rate dispersion of the (110) and (101) crystal faces was determined as a function of sodium chloride concentration, temperature, and solution pH. Under the conditions investigated, the growth rate dispersion follows the constant crystal growth model, in which each individual crystal is assumed to have a unique, constant growth rate. While the growth rate dispersion of the (110) face seems independent of the solution conditions, for the (101) face it was observed to vary systematically with temperature and pH. The greater susceptibility of the (101) face to the causes of growth rate dispersion was interpreted in light of a model proposed to explain the differing growth mechanisms of each face. Overall, the magnitude of crystal growth rate dispersion observed for lysozyme is similar to that reported for some small organic molecules.

Macromolecular crystallography at high pressure with pneumatic diamond anvil cells handled by a six-axis robotic arm

A new pneumatic diamond anvil cell has been constructed, generating continuous pressure and temperature variations in the range 0–2.5 GPa and 293–393 K. The cell is designed mainly for high-pressure macromolecular crystallography and should facilitate pressure and temperature annealing of the sample. The analysis is reported of several diffraction data sets of tetragonal hen egg-white lysozyme crystals loaded either in the new cell or in a currently used membrane-based cell. These experiments were performed on beamline FIP-BM30A at the ESRF, Grenoble, a macromolecular crystallography beamline on a bending magnet. Cells were handled and automatically centred by a six-axis robotic arm that was used as a goniometer for data collection by the oscillation method.

Role of the Electric Double Layer in Controlling the Nucleation Rate for Tetragonal Hen Egg White Lysozyme Crystals by Application of an External Electric Field

The nucleation rate of tetragonal hen egg white (HEW) lysozyme crystals could be increased or decreased via application of an ac electric field, which modifies the chemical potential of the liquid and solid states. The strength of an external electric field necessary to control the nucleation rate was calculated to be 104−105 V/cm, which was larger than that used experimentally by 2 or 3 orders of magnitude. This huge discrepancy could be understood by assuming the presence of a very large electric field sustained in the electric double layer (EDL) that forms at the interface between the dissimilar phases. By changing the frequency of an applied electric field of 800 V/cm in the range of 1−9 MHz, we were able to increase or decrease the nucleation rate. Specifically, the nucleation rate with an applied electric field increased with an increasing applied frequency up to 5 MHz and then significantly dropped above this value. The rapid decrease is considered to result from extinction of the EDL above 5 MHz.

Solubility measurements of protein crystals under high pressure by in situ observation of steps on crystal surfaces

We successfully measured equilibrium temperatures Te of tetragonal hen egg-white lysozyme crystals under high pressure by in situ observation of steps on the {110} faces of the crystals. The dependence of Te on the concentration of lysozyme corresponds to that of the solubility Ce on temperature. The precision of the solubility determined in this study is significantly higher than that in previous works. One Te could be measured during short time less than 70 minutes. This method for solubility measurements of protein crystals under high pressure is the fastest one at this stage.

Theory of the Solubility of Protein Crystals

We present a theory describing the solubility of protein crystals as a function of pH, salt concentration, and temperature. There are four terms in the model. The neutral terms arise from 1) the translational entropy of the soluble proteins, and 2) H-bond and hydrophobic attractive interactions which we obtain from a fitting procedure. The two electrostatic terms are a result of counterions confined in the crystal to satisfy charge neutrality. These counterions contribute 3) an entropic penalty from the trapping of ions in the crystal, and 4) a favorable enthalpy from the interaction of each protein with its counterion cloud. This theory quantitatively describes the solubility of tetragonal and orthorhombic lysozyme crystals as determined by Pusey et al. According to the theory, the reduced solubility at high salt concentrations comes, not from increased screening, but from a reduced entropy of counterion confinement. The theory correctly describes the weak pH dependence of the solubility, which is a result of the compensating effects of the two electrostatic terms. We discuss the implications of this theory for crystal nucleation and the success of the “crystallization slot”.

Modeling the Mechanical Response of Tetragonal Lysozyme Crystals

We investigate the temperature- and humidity-dependent mechanical response of tetragonal lysozyme crystals using a continuum-based crystal plasticity model calibrated with existing experimental data. The effects of temperature and intracrystalline water are incorporated in the elastic constant of the protein crystal as well as the critical resolved shear stress on the slip planes in the crystal plasticity model. The critical resolved shear stresses have been deduced from Vickers microhardness test data corresponding to different temperatures and states of hydration. Compression analyses have then been carried out along different crystallographic directions of lysozyme crystals which reveal that their mechanical response is highly anisotropic and orientation dependent, being purely elastic along the [110] direction but elastoplastic along the [100] and [212] directions. An interesting observation is that an increase in temperature and the amount of intracrystalline water molecules leads to a decrease in the critical resolved shear stress of the slip systems resulting in softening of the crystal. The analysis presented in this paper may be applied to the study of other protein crystal systems as well as their optimal design for biotechnological applications.

Phase transition in tetragonal hen egg-white lysozyme crystals

Lattice dynamics of tetragonal hen egg-white lysozyme crystals was studied by Brillouin light scattering and differential ac-nanocalorimetry with controlled relative humidity from 298 to 330 K. Temperature dependences of fast quasitransverse phonon velocity and integral intensity were found to exhibit anomalies in the vicinity of 306 K. An anomaly in the temperature behavior of heat capacity was also observed near this temperature. It is concluded that the anomalies point to a structural phase transition. Possible mechanisms of phase transition in lysozyme crystals are discussed.

Elastic constants in tetragonal hen egg-white lysozyme crystals containing large amount of water

Transverse sound velocity of cross-linked tetragonal hen egg-white (HEW) lysozyme crystals containing large amount of water in the crystal was measured using ultrasonic pulse-echo method. All elastic constants of cross-linked crystals were observed to be C11=C22=5.50 GPa, C12=4.33 GPa, C13=C23=3.94 GPa, C33=5.22 GPa, C44=C55=0.68 GPa, and C66=0.84 GPa, respectively. We found that the elastic constants of the cross-linked crystals are identical to those of the intrinsic ones without cross-linking. Moreover, we found that tetragonal HEW lysozyme crystals that enclose large amount of water show decreased elastic constants (softening). In particular, the shear elastic constants C44=C55 and C66 showed more softening effect comparing with other elastic components.

Direct Observation of Adsorption Sites of Protein Impurities and Their Effects on Step Advancement of Protein Crystals

We measured noninvasively step velocities of elementary two-dimensional (2D) islands on {110} faces of tetragonal lysozyme crystals, under various supersaturations, by laser confocal microscopy combined with differential interference contrast microscopy. We studied the correlation between the effects of protein impurities on the growth of elementary steps and their adsorption sites on a crystal surface, using three kinds of proteins: fluorescent-labeled lysozyme (F-lysozyme), covalently bonded dimers of lysozyme (dimer), and a 18 kDa polypeptide (18 kDa). These three protein impurities suppressed the advancement of the steps. However, they exhibited different supersaturation dependencies of the suppression of the step velocities. To clarify the cause of this difference, we observed in situ the adsorption sites of individual molecules of F-lysozyme and fluorescent-labeled dimer (F-dimer) on the crystal surface by single-molecule visualization. We found that F-lysozyme adsorbed preferentially on steps (i.e., kinks), whereas F-dimer adsorbed randomly on terraces. Taking into account the different adsorption sites of F-lysozyme and F-dimer, we could successfully explain the different effects of the impurities on the step velocities. These observations strongly suggest that 18 kDa also adsorbs randomly on terraces. Seikagaku lysozyme exhibited a complex effect that could not alone be explained by the two major impurities (dimer and 18 kDa) present in Seikagaku lysozyme, indicating that trace amounts of other impurities significantly affect the step advancement.

Assessment of biomolecular force fields for molecular dynamics simulations in a protein crystal

Different biomolecular force fields (OPLS-AA, AMBER03, and GROMOS96) in conjunction with SPC, SPC/E and TIP3P water models are assessed for molecular dynamics simulations in a tetragonal lysozyme crystal. The root mean square deviations for the C(a) atoms of lysozymes are about 0.1 to 0.2 nm from OPLS-AA and AMBER03, smaller than 0.4 nm from GROMOS96. All force fields exhibit similar pattern in B-factors, whereas OPLS-AA and AMBER03 accurately reproduce experimental measurements. Despite slight variations, the primary secondary structures are well conserved using different force fields. Water diffusion in the crystal is approximately ten-fold slower than in bulk phase. The directional and average water diffusivities from OPLS-AA and AMBER03 along with SPC/E model match fairly well with experimental data. Compared to GROMOS96, OPLS-AA and AMBER03 predict larger hydrophilic solvent-accessible surface area of lysozyme, more hydrogen bonds between lysozyme and water, and higher percentage of water in hydration shell. SPC, SPC/E and TIP3P water models have similar performance in most energetic and structural properties, but SPC/E outperforms in water diffusion. While all force fields overestimate the mobility and electrical conductivity of NaCl, a combination of OPLS-AA for lysozyme and the Kirkwood-Buff model for ions is superior to others. As attributed to the steric restraints and surface interactions, the mobility and conductivity in the crystal are reduced by one to two orders of magnitude from aqueous solution.

Effects of a Magnetic Field on Lysozyme Crystal Nucleation and Growth in a Diffusive Environment

Tetragonal hen egg white lysozyme crystals were grown in agarose gel under the influence of a 7 T homogeneous and static magnetic field. The use of agarose, gels, and polymer solutions avoids crystal sedimentation and buoyancy-driven convection providing a diffusive mass transport environment. Under these conditions, 100% of lysozyme crystals grown in 0.02% (w/v) agarose can be oriented by a 7 T magnetic field. When the agarose concentration was increased, the number of oriented crystals decreased. The results are explained by the compromise between the effect of the magnetic field and the chemical effects due to the interaction between protein and agarose, because it is known that agarose modifies the nucleation rate of lysozyme crystals. When the chemical effect of agarose was removed, it appears clear that a homogeneous and constant magnetic field influences both nucleation and growth of lysozyme tetragonal crystals in a convection-free environment. Both effects depend on supersaturation and exposure time.

Control of Nucleation Rate for Tetragonal Hen-Egg White Lysozyme Crystals by Application of an Electric Field with Variable Frequencies

Control of the nucleation rate is important for the growth of high quality protein crystals; therefore, the development of improved growth methods is required. Crystal growth with an external electric field is an attractive method for control of the nucleation rate. However, mainly a decrease in nucleation rate was observed when an external electric field was applied using DC voltage. By focusing on the chemical potential of the liquid and solid, control of both the increase and the decrease in nucleation rate was successfully achieved by applying an AC electric field. Control of nucleation rate in terms of enhancement and retardation is reported for hen-egg white lysozyme crystals by imposition of an electric field with varying frequencies, which was determined by the electrical permittivity of both the hen-egg white lysozyme solution and crystals.