Crystallization of Chicken Egg White Lysozyme from Assorted Sulfate Salts
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Journal of Crystal Growth 196 (1999) 332—343 Crystallization of chicken egg white lysozyme from assorted sulfate salts Elizabeth L. Forsythe!, Edward H. Snell", Christine C. Malone!, Marc L. Pusey# * ! USRA, 4950 Corporate Drive, Suite 100, Huntsville, AL 35806, USA " NAS/NRC Associate, Biophysics ES76, NASA/MSFC, Huntsville, AL 35812, USA # Biophysics ES76, NASA/MSFC, Huntsville, AL 35812, USA Abstract Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Crystals have also been grown at 4°C in the absence of any other added salts using isoionic lysozyme which was titrated to pH 4.6 with dilute sulfuric acid. Four different crystal forms have been obtained, depending upon the temperature, protein concentra- tion, and precipitating salt employed. Crystals grown at 15°C were generally tetragonal, with space group P422. Crystallization at 20°C typically resulted in the formation of orthorhombic crystals, space group P222. The tetragonal % orthorhombic transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20°C and between 100 and 125 mg/ml protein concentration. Crystallization from 1.2 M magnesium " " " c" > sulfate at pH 7.8 gave a trigonal crystal, space group P32, a b 87.4, c 73.7, 120°, which diffracted to 2.8 A. Crystallization from ammonium sulfate at pH 4.6, generally at lower temperatures, was also found to result in a monoclinic form, space group C2, a"65.6, b"95.0, c"41.2, b"119.2°. A crystal of &0.2;0.2;0.5 mm grown from bulk solution diffracted to &3.5 A> . ( 1999 Elsevier Science B.V. All rights reserved. Keywords: Lysozyme; Sulfates; Trigonal; Monoclinic; Crystallization 1. Introduction ions are the dominant precipitating species for de- termining CEWL solubility and crystal space Chicken egg white lysozyme (CEWL) is the most group [1]. It has been shown that phosphate, acet- common model protein for macromolecular crystal ate, carbonate, chloride, bromide, citrate, nitrate, growth studies. Previous work has shown that an- iodide, and thiocyanate anions, as well as ethanol and sodium para-toluenesulfonate can crystallize CEWL [1—8]. Sulfates, particularly ammonium sulfate, are one * Corresponding author. Fax: #1 256 544 6660; e-mail: of the most commonly employed precipitants for [email protected]. protein crystallization [9,10]. While the ability to 0022-0248/99/$ — see front matter ( 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 2 4 8 ( 9 8 ) 0 0 8 4 3 - 4 E.L. Forsythe et al. / Journal of Crystal Growth 196 (1999) 332–343 333 grow crystals from sulfate salts was mentioned in 2. Materials and methods the earliest report of lysozyme crystallization [11], native lysozyme was believed to only give amorph- CEWL (Sigma) was repurified by cation ex- ous precipitates from sulfate salts. Indeed, the sec- change chromatography and recrystallized as pre- ond report on lysozyme crystallization [2] stated viously described [21]. The recrystallized protein that while their “first crystalline lysozyme prepara- was then dialyzed against several changes of distil- tion was obtained from an acetate buffer on addition led water, then concentrated using an Amicon of ammonium sulfate” they had “never been success- YM-3 membrane to the desired final concentration. ful in obtaining crystalline material from a system Completely desalted, or isoionic lysozyme, was pre- which contained only the sulfate or the acetate ani- pared by the method of Rie`s-Kautt et al. [17]. All on.” This supposed inability to crystallize from sul- chemicals were reagent grade or better. All buffers fate salts has been the basis for several comparative were prepared by making a solution of&95% final studies of crystal versus amorphous precipitate volume containing the desired final molar amount forming conditions [12—15]. Steinrauf [3] obtained of the buffering species, adding salt to the desired crystals using 10% (0.7 M) sodium sulfate, but in final concentration, then titrating the buffer with the presence of 1.0 M acetate buffer. Orthorhombic the counter ion acid or base to the desired pH. lysozyme crystals were obtained from ammonium Once the pH was obtained the volume was ad- sulfate at basic pH, but this was after all lysine side justed to the final value with dHO. Crystal- chain amines had been reductively methylated lizations were performed using the sitting drop [16]. Rie`s-Kautt et al. [17] have shown that purifi- technique [22] using a 1 : 1 mixture of protein in ed isoionic lysozyme can be crystallized from distilled water and reservoir solution (10 ll of each stoichiometric amounts of sulfate at basic pH. They solution) for the drop. Several different protein also reported that crystals could not be obtained at preparations were used over the course of these acidic pH. Broide et al. [18] showed that protein experiments. Unless otherwise noted the pH values straight from the bottle (not further purified) gave are those of the buffered precipitant (reservoir) tetragonal crystals from magnesium, potassium, solution prior to mixing with the protein. The sit- and ammonium sulfates at pH 7.8. Vuillard et al. ting drops were kept in incubators controlled to the [19] showed that a novel triclinic form of CEWL set point $0.5°. Concentrations refer to those of could be batch crystallized from 75 mg/ml ly- the starting solution components prior to mixing. sozyme, 22% (&0.9 M) ammonium sulfate at pH In theory, these are also the final values assum- 4.6 in the presence of 0.5 M dimethyl ethylam- ing the 1 : 1 mixture undergoes a 50% reduction monium propane sulfonate, a zwitterionic solu- in volume over the course of the equilibration bilizing agent. process. We have recently re-examined the use of sulfates Suitable crystals were mounted in capillaries for as a crystallization agent for CEWL and reported crystallographic analysis. X-rays were produced on the sitting drop crystallization of CEWL using from a Rigaku rotating anode source operated at ammonium sulfate from pH 4.0 to 7.8 [20]. It was 40 kV (70 mA) with a fine 300 lm focus and 300 lm found that high protein and low ammonium sulfate collimator and CuKa radiation at 1.54 A> . Data concentrations were key to successful crystalliza- were collected at room temperature using an R-axis tion. Herein, we extend our findings with a report of II image plate with 105 lm pixel scan. The X-ray the crystallization of CEWL from a variety of sul- diffraction data were processed with the programs fates over the same pH range, the effects of temper- Denzo and Scalepack [23]. ature and protein concentration on the crystal form Solubility measurements were made using the obtained, the growth of CEWL crystals using microcolumn method developed in this laboratory isoionic protein titrated to acidic pH using dilute [24,25]. Crystals used to pack the columns were sulfuric acid, and the growth of trigonal and mono- prepared by dialysis of an &150 mg/ml lysozyme clinic crystal forms of CEWL from buffered sulfate solution against 0.1 M sodium phosphate, 0.3 M salts. ammonium sulfate, pH 6.8, in a thermostatted 334 E.L. Forsythe et al. / Journal of Crystal Growth 196 (1999) 332–343 beaker at 10°C. A portion of the crystals and super- tained. The 15°C sitting drops gave both tetragonal natant solution in the dialysis bag was removed, and orthorhombic crystals, while those at 20°C and the remainder dialyzed against 0.1 M sodium gave orthorhombic. phosphate, 0.4 M ammonium sulfate. About of Table 3 summarizes the diffraction data for the the crystals and supernatant from this dialysis was CEWL crystals obtained from the different sulfate removed, and the remainder dialyzed against 0.1 M salts. Generally, only small crystals were used to sodium phosphate, 0.6 M ammonium sulfate. The collect the orthorhombic data, due to their tend- crystals and supernatant solutions from these suc- ency to form a rather dense mass. Orthorhombic cessive dialyses were used to pack and equilibrate crystals (*0.3—0.4 mm) were somewhat fragile, three sets of columns for solubility determination, tended to be cracked, and difficult to separate from with the supernatant solutions from the three dialy- other crystals. Manganese sulfate is the only salt sis operations being used as stock material for which so far has not given tetragonal crystals. Crys- preparation of the respective solubility reservoir tallization experiments were not attempted in the solutions. pH 6.0 region with manganese sulfate due to the formation of a brown precipitate. Also, both mag- nesium and manganese sulfates gave crystals to 3. Results higher (1.2 M and 1.7 M, respectively) salt concen- trations than the monovalent cation salts. The unit 3.1. Crystallization from different sulfate salts cell dimensions for the tetragonal crystals ranged from a"b"78.6—78.7 A> , c"38.6 A> , close to the An initial series of sitting drop crystallization dimensions previously reported for this morpho- trials using a variety of sulfate salts were made at logy, a"b"78.8—79.2 A> , c"37.9—38.5 A> [1,3,17, room temperature, which typically varied from 26—29]. Whether the variations are due to salt- &17 to 24°C. The results from these experiments induced alterations in the dimensions or to general then led to subsequent experiments in incubators variations in the unit cell remains to be determined. with the temperatures controlled to $0.5°Cor The orthorhombic unit cell dimensions for crystals better.