Marc L. Pusey

The solubility of the tetragonal form of hen egg white lysozyme from pH 4.0 to 5.4

Abstract Hen egg white lysozyme solubilities in the presence of the tetragonal crystal form have been determined. Conditions investigated cover the pH range 4.0 to 5.4, varying from 2.0% to 7.0% NaCl concentrations and from 4 to 25°C. In all instances, the solubilities were found to increase with temperature and decrease with increasing salt concentration. The effect of pH were more complex, showing a decreasing solubility with increasing pH at low salt concentration and an increasing solubility with increasing pH at high salt concentration.

Growth kinetics of tetragonal lysozyme crystals

Abstract A method has been devised for immobilizing protein crystals in small volumes under defined conditions in order to determine growth rates on various faces. Using this method, we have investigated the growth kinetics of the [110] face of tetragonal hens egg white lysozyme crystals at varying degrees of bulk supersaturation. The growth rate data were analyzed using a simple convective-diffusive model to determine an empirical relationship between growth rate and local supersaturation at the interface. This empirical relationship describes the surface kinetics of the growth process, which together with the convective-diffusive model can be used to predict various details of the growth process as a function of crystal size, bulk supersaturation, and gravity level. It was shown that transport is dominated by convection in normal gravity for all crystal sizes larger than a few microns whereas transport is diffusion limited in a microgravity environment for sizes up to a few millimeters. Convection can become significant even in a microgravity environment for crystals approaching cm sizes. Growth of lysozyme will always be limited by surface kinetics in normal gravity because convective transport is sufficient to supply solute to the growth interface as fast as it can be incorporated into the lattice. In microgravity the transport can become sufficiently slow as the crystal becomes larger so that growth is limited by transport rather than surface kinetics. The implications of this are discussed.

Preliminary investigations into solutal flow about growing tetragonal lysozyme crystals

Abstract A series of preliminary experiments were done to investigate solutal flow about growing lysozyme crystals and its effects. Density gradient driven flow as observed using a schlieren optical system. Crystals used ranged from 0.3 to 1.72 mm across the (110) face, and protein concentrations were from 3.7 to 23.7 mg/ml ( C sat = 1.2 mg/ml at 18°C). The convective plume velocities were found to be from 10 to 50 μm/s, which correlated with those predicted to occur based upon a diffusive-convective model. When micro-crystals of lysozyme ( .5 mm) crystals in the terminal phases of a typical crystal growth procedure.

The promise of macromolecular crystallization in microfluidic chips

Microfluidics, or lab-on-a-chip technology, is proving to be a powerful, rapid, and efficient approach to a wide variety of bioanalytical and microscale biopreparative needs. The low materials consumption, combined with the potential for packing a large number of experiments in a few cubic centimeters, makes it an attractive technique for both initial screening and subsequent optimization of macromolecular crystallization conditions. Screening operations, which require a macromolecule solution with a standard set of premixed solutions, are relatively straightforward and have been successfully demonstrated in a microfluidics platform. Optimization methods, in which crystallization solutions are independently formulated from a range of stock solutions, are considerably more complex and have yet to be demonstrated. To be competitive with either approach, a microfluidics system must offer ease of operation, be able to maintain a sealed environment over several weeks to months, and give ready access for the observation and harvesting of crystals as they are grown.

Protein solubilities determined by a rapid technique and modification of that technique to a micro-method

Abstract This laboratory has developed a simple, rapid method for determination of protein solubilities [Pusey and Gernert, J. Crystal Growth 88 (1988) 419]. The system was based upon maximization of the available crystalline surface area and minimization of the free solution volume to be brought into equilibrium. We have determined the tetragonal lysozyme solubility diagram from pH 4.0 to 5.2 (0.1 M sodium acetate), 2%–7% NaCl, 3–25°C, and portions of the orthorhombic solubility diagram using this technique. Both the tetragonal and orthorhombic solubilities were found to increase smoothly with decreasing salt concentration and increasing temperature. No retrograde solubilities were observed. A major drawback was a requirement for 1–5 mL (ca. 0.5 to 2.5 g) of crystalline protein per column, leading to the devising of micro-methods for implementing this technique. Using column volumes of 75, 300, and 900 μL, identical tetragonal lysozyme solubility diagrams (pH 4.5, 0.1M sodium acetate, 5–25°C) were obtained. Chymotrypsinogen solubilities have also been determined using this apparatus, being retrograde over the temperature range tested. Currently, the primary limiting factor in reducing the crystalline volume is the minimum solution sample size needed to accurately quantitate the protein.

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.

The effects of temperature and NaCl concentration on tetragonal lysozyme face growth rates

Measurements were made of the (110) and (101) face growth rates of the tetragonal form of hen egg white lysozyme at 0.1M sodium acetate buffer, pH 4.0, from 4 to 22°C and with 3.0%, 5.0%, and 7.0% NaCl used as the precipitating salt. The data were collected at supersaturation ratios ranging from ∼4 to ∼63. Both decreasing temperature and increasing salt concentrations shifted plots of the growth rate versus C/Csat to the right, i.e. higher supersaturations were required for comparable growth rates. The observed trends in the growth data are counter to those expected from the solubility data. If tetragonal lysozyme crystal growth is by addition of ordered aggregates from the solution, then the observed growth data could be explained as a result of the effects of lowered temperature and increased salt concentration on the kinetics and equilibrium processes governing protein-protein interactions in solution. The data indicate that temperature would be a more tractable means of controlling the growth rate for tetragonal lysozyme crystals contrary to the usual practice in, e.g., vapor diffusion protein crystal growth, where both the precipitant and protein concentrations are simultaneously increased. However, the available range for control is dependent upon the protein concentration, with the greatest growth rate control being at the lower concentration.

The effect of protein impurities on lysozyme crystal growth

While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. To further examine the issue of purity in macromolecule crystallization, this study investigates the effect of the protein impurities, avidin, ovalbumin, and conalbumin at concentrations up to 50%, on the solubility, crystal face growth rates, and crystal purity of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the ¿110¿ and ¿101¿ lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification.

The Averaged Face Growth Rates of lysozyme Crystals: The Effect of Temperature

Abstract Measurements of the averaged or macroscopic face growth rates of lysozyme crystals are reported here for the (110) face of tetragonal lysozyme, at three sets of pH and salt concentrations, with temperatures over a 4–22°C range for several protein concentrations. The growth rate trends with supersaturation were similar to previous microscopic growth rate measurements. However, it was found that at high supersaturations the growth rates attain a maximum and then start decreasing. No “dead zone” was observed but the growth rates were found to approach zero asymptotically at very low supersaturations. The growth rate data also displayed a dependence on pH and salt concentration which could not be characterized solely by the supersaturation. A complete mechanism for lysozyme crystal growth, involving the formation of an aggregate growth unit, mass transport of the growth unit to the crystal interface and faceted crystal growth by growth unit addition, is suggested. Such a mechanism may provide a more consistent explanation for the observed growth rate trends than those suggested by other investigators. The nutrient solution interactions leading to the formation of the aggregate growth unit may, thus, be as important as those occurring at the crystal interface and may account for the differences between small molecule and protein crystal growth.

Estimation of the initial equilibrium constants in the formation of tetragonal lysozyme nuclei

Abstract Knowledge of the solution aggregate composition in both undersaturated (pre-nucleated) and oversaturated (nucleated and crystal growing) solutions is important to the eventual understanding of the (protein) crystal nucleation and growth process. Using relative light scattering intensity measurements at 90δ, estimates of the initial equilibrium constants in the formation of tetragonal lysozyme nuclei have been made. While the aggregation pathway is not known, it is assumed that the first step is dimer formation ( K 1 = [P 2 ]/[P 1 ] 2 ). Experiments at 3.0% NaCl, 0.1M acetate buffer, pH 4.0, 22°C indicate K 1 values of (1−3) × 10 4 L mol -1 . Estimation of subsequent equilibrium constants will be dependent upon the aggregation model chosen or determined. For a 1 → 2 → 4 pathway, K 2 values are estimated to be (2−4) × 10 3 L mol -1 , while for a 1 → 2 → 3 pathway, values of (10−20) × 1 0 3 L mol -1 are estimated. Equilibrium values of this magnitude essentially “fix” the monomer concentration by the saturation concentration point, with only the higher order aggregates showing appreciable concentration increase with increasing total protein concentration. This strongly suggests that tetragonal lysozyme crystal growth is by addition of aggregates preformed in the bulk solution, not by monomer addition. This is independent of a screw dislocation or surface nucleation type mechanism.