Temperature-dependent solubility of selected proteins

Journal of Crystal Growth 191 (1998) 820—826

Temperature-dependent solubility of selected proteins G.K. Christopher!,*, A.G. Phipps!, R.J. Gray" ! Diversified Scientific, Inc., 2800 Milan Ct., Suite 381, Birmingham, AL 35211-6908, USA " Center for Macromolecular Crystallography, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA Received 25 November 1997; accepted 3 February 1998

Abstract Temperature is being recognized as a noninvasive control parameter for protein crystallization. Before temperatureinduced crystallization can be routinely used as a method of preparation of protein crystals, qualitative data on the temperature dependent solubility of the protein must be obtained. Qualitative data for temperature-dependent solubility is available for a limited number of proteins. We report herein qualitative temperature-dependent solubility data for selected proteins as obtained by the use of a multichambered thermal gradient device. These studies demonstrate that a large percentage of proteins do in fact exhibit a solubility versus temperature dependence which suggests that temperature can be used as an alternative crystallization technique for proteins. ( 1998 Elsevier Science B.V. All rights reserved. Keywords: Solubility; Proteins; Temperature

1. Introduction Temperature as a control parameter for protein crystal growth has attracted considerable interest of late both on the ground and in microgravity [1—8]. Exploiting temperature control of protein crystal growth offers several advantages. The chief advantage of a temperature driven crystallization experiment is that it provides rapid, precise and reversible control of the supersaturation level.

* Corresponding author. Fax: #1 205 943 6612; e-mail: [email protected].

One of the authors has been investigating the applicability of dynamic temperature control systems to provide protein crystals for X-ray analyses. In order to use temperature as a controlling variable in crystallization of proteins, qualitative data on the temperature dependent solubility of the protein is necessary. These data are not readily available in the literature. These studies were undertaken to ascertain the general utility of the temperature-based crystallization systems. As detailed in the following paragraphs, studies utilizing a multichambered thermal gradient device demonstrate that a large percentage of proteins do in fact exhibit a solubility versus temperature dependence. This is significant in that it suggests that temperature can be used as an alternative

0022-0248/98/$19.00 ( 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 0 2 4 8 ( 9 8 ) 0 0 3 5 5 - 8

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crystallization technique for a large percentage of proteins. This is important to the crystallographic community for several reasons: 1. The use of an alternative crystallization technique increases the probability of producing crystals of new, previously uncrystallized proteins. 2. Temperature is amenable to control, thereby allowing precise control of the approach to nucleation and post nucleation crystal growth. 3. Temperature control is also noninvasive and does not require manipulation of the crystallization solution. 4. Previously crystallized proteins are potential candidates for temperature-induced crystallization. It is possible that temperature crystallization of proteins will provide larger and/ or higher quality crystals that could result in enhanced X-ray diffraction data and thus higher resolution structures. 2. Materials and methods Thirty commercially available randomly chosen proteins were tested for temperature dependent solubility in a multichambered thermal gradient

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device (T-Block) developed at the University of Alabama at Birmingham (UAB) [6,7]. This device allows for the maintenance of a temperature gradient of 25—30°C across an aluminum block. A 4—45°C range was obtained by altering water bath temperatures and via the use of a standard refrigerator. Samples were loaded into glass microcapillaries and placed at intervals corresponding to 1—2°C temperature difference. The experimental set up is shown in Fig. 1 with the T-block located to the left of the manual optics version of the CrystalScoreTM image acquisition and archiving system. Temperature of the T-block was maintained by two water baths. After a defined incubation period, normally two days, microcapillaries were taken from the T-block and microscopically examined over their length for protein crystals or precipitate using the CrystalScoreTM system. Images and experimental conditions were saved to a database for later analysis of solubility behavior over the temperature gradient. Proteins were chosen for analysis based upon criteria including (1) previous crystallization using a method adaptable to microbatch; (2) commercial availability and (3) ability to purchase economically. Table 1 shows the proteins that were used in

Fig. 1. Apparatus used in protein screening.

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Table 1 Summary of proteins tested for temperature dependent solubility and experimental conditions Protein

MW approx (K)

Source

A1¤ 1 #. 280 nm

Protein buffer and preparation temperature

Mix 1 : 1 with

Elastase Papain

26 23

Pig/pancreas Papaya

20.2 25

NA NA

Ribonuclease A Trypsin Rennin AP

14 24 31 89

Bovine/pancreas 6.95 Pig/pancreas 16.6 Bovine/calf stomach 14.3 E. coli 7.2

0.01 M NaAc, pH 5.0, cold prep Water, cold, add+25%v/v aq. NaCl 25%, filter 0.45 l, cold prep 25 mM NaAc, pH 7.0, cold prep 0.2 M Tris, pH 8.3 0..025 M NaCitrate, ph 5.5 0.05 M Tris, 0.01 M MgCl 2

Pepsin BSA HEA (ovalbumin)

57 34 67 45

Bovine intestine Porcine/stomach Bovine Chicken

10 14.1 6.61 7.5

pH 8.0, cold prep 0.25 M HCl, pH 2.0 25 mM NaAc, pH 5.0 cold prep 100 mM NaCit, pH 5.0, cold prep

ESA WGA

66 19

Horse T. vulgaris

4.04 10.9

Cellulase Thaumatin

46 21

T. viride T. daniellii

25 7.6

50 mM NaAc pH 5.6, cold prep 0.01 M NaAc, pH 4.7 containing 0.006 M CaCl 2 0.02 M Tris, pH 8.3, Water

Insulin-human Concanavalin A Catalase Edestin Lysozyme a-Amylase b-Amylase a-Lactalbumin apo-Transferrin

5.7 27 240 50 c 14.4 55 59 14 75

Recombinant Jack bean Bovine/liver Hemp Chicken/egg white Bacillus Barley Bovine/milk Bovine

10.4 11.4 36.5 7c 26.04 25.3 17.7sp 20.9 14

See ! 0.01 M PB, pH 7.4, 0.45 filtered 0.1 M PB, pH 7.4, 0.45 filtered 10% AS 50 mM NaAc, pH 4.4 25 mM NaAc, pH 4, cold prep 25 mM NaAc, pH4, cold prep 0.1 M Tris, pH 6.5, cold prep 0.01 M PB, pH 7.4

a-Chymotysinogen 25

Bovine/pancreas

20.6

IgG Ubiquitin Ribonuclease B Glucose isomerase Fibrinogen Pepsinogen

150 8.5 14 157

Bovine 13.44 Bovine/erythrocyte 1.75 c Bovine pancreas 6.95 Strep. rubiginosus 10

0.142 M Na HPO , 0.129 M citric 2 4 acid, 4% EtOH, cold prep 0.01 M PB, pH 7.4, cold prep 0.05 M cacodylate, pH 5.5, cold prep 25 mM NaAc, pH 7, cold prep 50 mM Tris, pH 8

2 M AS in buffer 30% PEG 4000 in buffer 75% sat AS in buffer 1 : 2 w/3.6 M AS in buffer

250 39

Bovine plasma Pig/stomach

10 mM MES, pH 6.2 0.1 M PB, pH 6.25

1% AS in same buffer 80% sat AS in same buffer

15.6 13.05

75% sat A.S in same buffer 25% AS in same buffer 15% sat AS in same buffer 1 : 2 w/56% sat AS in same buffer NA Sat AS in same buffer Mix with sat AS in same buffer dropwise until cloudy, filter 0.45 l 51% sat AS in same buffer NA 30% PEG 8000 in buffer 0.1 M ADA, pH 6.5 0.75 M Naktartrate NA NA NA 10% NaCl in buffer 60% sat As in buffer 60% sat AS in buffer sat As in buffer 20%PEG 4000 in same buffer 45% sat As in same buffer

Note: A1¤ from Ref. [16]. 1#. c"calculated molecular absorption coefficient based on amino acid sequence [9]. sp"sweet potato, used as the closest approximation available. !To 40 mg h-insulin-zinc (Eli Lilly, Indianapolis, IN) was added in sequence: 4 ml of 0.02 M HCl, 0.4 ml 0.15 M ZnAcetate, 2.0 ml of 0.2 M NaCitrate, 1.33 ml acetone, 0.26 ml H O, after which pH was adjusted with 1.0 M NaOH to 10.6 and lowered to 6.39 with HCl 2 and placed in a 50°C water bath to clear. Temperature of the water bath was decreased to 44°C and the sample allowed to equilibrate before loading into capillary tubes. The following abbreviations apply to all tables: NA"not applicable, ESA"equine serum albumin, BSA"bovine serum albumin, WGA"wheat germ agglutinin, PEG"polyethylene glycol, AS"ammonium sulfate, PB"phosphate buffer, NaAc"sodium acetate, NaCit"sodium citrate, NaKT"sodium potassium tartrate, Tris"tris (hydroxymethyl) aminomethane, MES"2-(4-morpholino)-ethane sulfonic acid, EtOH"ethanol, IgG"immunoglobulin G, Con A"concanavalin A, AP"alkaline phosphatase, ADA"N-[2-acetamido]-2-iminodiacetic acid.

G.K. Christopher et al. / Journal of Crystal Growth 191 (1998) 820–826

this study and the experimental conditions. All proteins, except concanavalin A (Seikagaku), glucose isomerase and human insulin, were purchased from Sigma Chemical Co. (St. Louis, MO). Proteins, with the exception of equine serum albumin, which was purified by size exclusion chromatography, and E. coli alkaline phosphatase, which was dialyzed and concentrated prior to use, were used as received without further purification. All other reagents were purchased from Fisher Scientific or Sigma Chemical Co. In general, proteins were dissolved in buffer and filtered using a 0.2 l cellulose acetate syringe filter prior to use. Protein concentration was calculated by determining absorbance at 280 nm. Growth media were mixed, drawn into microcapillaries and then sealed. Temperature of the preparation proved to be important in many cases as noted in Table 1.

3. Results and discussion Table 2 summarizes the results obtained for the 30 proteins tested for temperature dependent solubility characteristics. Of the 30 proteins tested as set forth in Table 1, 28 (93%) provided interpretable results. Of those 28 proteins, 24 (86%) demonstrated a temperature-dependent solubility. Of those 24 proteins, 13 (54%) demonstrated a retrograde solubility (meaning a higher solubility at lower temperatures; these proteins were typically crystallized using divalent anions as the precipitating agent). Table 3 gives more detail on the final growth conditions and crystallization range as determined in this study. Crystallization ranges are given as the best average of at least three trials. Quantity, size and quality of crystals were influenced by temperature as well. This is due either to the rate at which the sample changes temperature once it is placed in the block, or to the final equilibrium temperature (or to a combination of both these variables). Fig. 2 shows selected images of crystals obtained using the above crystallization conditions, the CrystalScoreTM software/instrumentation and polarized light. Growth temperature was 32° for wheat germ agglutinin, 16°C for thaumatin, 8°C for IgG, 13.7°C for lysozyme, 5.7°C

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Table 2 T-block temperature-dependent solubility and crystallization results Proteins with temperature-dependent solubility f Lysozyme f Thaumatin f Cellulase f Concanavalin A (Con A)! f Bovine serum albumen (BSA) f Equine serum albumen (ESA)! f Ovalbumin (HEA)! f Pepsin f Edestin f a-Amylase! f b-Amylase! f Trypsin f Alkaline phosphatase, bovine intestinal mucosa! f Catalase! f Glucose isomerase! f Elastase! f a-Chymotrypsinogen! f Papain! f apo-Transferrin f Alkaline phosphatase, E. coli! f Fibrinogen f Human insulin f Ribonuclease B f a-Lactalbumen! Proteins that did not exhibit a temperature-dependent solubility f Wheat germ agglutinin (WGA) f Bovine IgG f Ubiquitin f Rennin Proteins that provided inconclusive results f Ribonuclease A f Pepsinogen !Retrograde solubility suggested.

for concanavalin A, and 20°C for cellulase. Final growth conditions are described in Table 3. Crystals of lysozyme, human insulin, bovine insulin, porcine insulin, ESA and neurophysindipeptide complex and other proteins have been grown by appropriate temperature changes [6,7]. This is the first comprehensive report of temperature-dependent solubility data for such a large number of proteins. Analysis of these 30 randomly chosen proteins, of which 86% demonstrated a temperature dependent solubility, suggests that temperature induced crystallization of proteins

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Table 3 Final growth conditions and crystallization results for proteins providing interpretable results Protein

Final growth conditions

Crystallization range

Lysozyme

24—35 mg/ml in 50 mM NaAc pH 4.4 w/5% NaCl 58—73 mg/ml in 0.38 M NaKT 0.05 M ADA pH 6.5 33—43 mg/ml in 0.02 M Tris 15% PEG 8000 pH 8.3

Crystals from 4 to 22°C, best crystals observed from 12 to 18°C Crystals from 4 to 28°C, best crystals observed from 14 to 23°C at 60 mg/ml Precipitate from 4 to 11°C, precipitate and crystals from 12 to 19%, and crystals only from 20 to 43°C best observed from 33 to 43°C Precipitate from 8 to 17°C, crystals from 18 to 37°C

Thaumatin Cellulase

BSA Pepsin Edestin

52—63 mg/ml in 25 mM NaAc w/38—50% sat A.S. pH 4.9 26—37 mg/ml in 0.25 M HCl pH 2.0 98—140 mg/ml in 10% A.S. Heat to 50° for 1 h

Con A

36 mg/ml in 100 mM PB buffer pH 7.4

a-Amylase

5—9 mg/ml in 25 mM NaAc pH 4.0 w/30% sat A.S. 4—5 mg/ml in 25 mM NaAc pH 4.0 w/30% sat A.S. 29—43 mg/ml in 0.2 M Tris pH 8.3 w/12.5% sat A.S.

b-Amylase Trypsin

Ribonuclease A

Catalase

45—54 mg/ml in 25 mM NaAc pH 7.0 w/38% sat A.S. 33 mg/ml in 0.05 M Tris, 0.01 M MgCl 2 pH 8.0, 37% sat A.S. 75 mg/ml in 100 mM PB, pH 7.4

Glucose isomerase ESA (purified)

(100 ll) 50 mM Tris, pH 8.0, 1.8 M A.S. 25 mg/ml in 50 mM NaAc pH 5.6 51% sat A.S.

WGA

10—13 mg/ml in 0.01 M NaAc pH 4.7, 3 mM CaCl 2 17 mg/ml in 0.1 M NaAc pH 5.0 0.142 M Na HPO , 0.129 M citric acid, 2 4 4% EtOH, 23% sat A.S. 60 mg/ml 100 mM NaCitrate pH 5.0 mixed w/ sat A.S. 45 mg/ml in H O with 5% NaCl 2

AP (b-int. m)

Elastase a-Chymotrypsinogen HEA Papain apo-Transferrin Human insulin Fibrinogen AP-E. coli Rennin Ribonuclease B IgG-bovine Ubiquitin a-Lactalbumen Pepsinogen

17—21 mg/ml in 0.001 M PB, pH 7.4 10% PEG 4000 (See d1 below) 24—27 mg/ml in 10 mM MES 0.5% A.S. pH 6.2 10 mg/ml in 0.05 mM Tris, 0.01 M MgCl , 2 pH 8.0, 50% sat A.S. 7—9 mg/ml in 25 mM NaCitrate pH 5.5 7.5% sat A.S. 30—54 mg/ml in 25 mM NaAc, pH 7, 37% sat A.S. 12.5 mg/ml in 0.01 M PB, pH 7.4, 1 M A.S. 12 mg/ml in 0.05 M cacodylate, pH 5.5, 15% PEG 7 mg/ml in 0.1 M Tris, pH 6.5, 50% sat A.S. 25 mg/ml in 0.1 M PB, pH 6.25, 40% sat A.S.

Note: Unless otherwise noted, results reflect two days of growth.

crystals from 4 to 26°C Precipitate from 10 to 16°C, showers from 16 to 21°C, crystals from 22 to 30°C Soluble from 4 to 15°C and precipitate from 15 to 43°C Crystals and precipitate from 4 to 17°C. Precipitate only above 18°C Crystals from 4 to 16°C, precipitate and crystals from 17 to 27°C, precipitate only above 27°C Crystals from 4 to 28°C, precipitate and crystals from 28 to 39°C, precipitate only at 41° and clear after 41°C Inconclusive, suggestive of crystallization range 8 to 20°C, occassionally at 4°C and increases to 20°C Crystals from 4 to 23°C Soluble to 14°C, crystals from 15 to 23°C, crystals and precipitate from 25 to 43°C Crystals from 4 to 18°C after 5 days, soluble after 18° Soluble from 4 to 14°C, Crystals from 15 to 43°C after 24 h Crystallizes throughout range under these conditions. Non-temperature dependent Crystals from 4 to 19°C after 1 1/2 h Crystals form 4 to 19°C Soluble from 4 to 15°C, precipitate after 15°C Soluble from 4 to 10°C, crystals at 12°C, precipitate from 14 to 43°C Crystals from 5 to 25°C, soluble after 26°C Crystals from 22 to 35°C, precipitate from 36 to 40°C Crystallizes from 4 to 21°C, soluble after 22°C Crystallizes from 4 to 19°C, soluble after 20°C Crystallizes throughout range under these conditions. Nontemperature dependent Crystallizes from 4 to 27°C Crystallizes throughout range, not temperature dependent Crystallizes throughout range, not temperature dependent Crystallizes 4 to 28°C, precipitate thereafter to 40°C Inconclusive

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Fig. 2. Selected crystal images from T-block experiments.

could be a generally useful technique. Interestingly, 50% of the proteins were more soluble at lower temperatures. Our data are consistent with those of similar studies [7,14,15] in percentages and individual protein solubility data. The multichambered thermal gradient device developed at UAB is useful as a rapid qualitative screening method for obtaining protein solubility data.

4. Summary As a crystallization technique, temperature is both noninvasive and easily and precisely controllable. When a large number of protein crystals are desired and sufficient quantities of protein are available, temperature induced crystallization can be the method of choice [8,10—13]. The actual quantity of protein necessary to obtain solubility data in the T-block is dependent upon the particular protein, but, at worst, should be 60—70 mg. Some proteins display a characteristic increased solubility with increasing temperature, while others display a decreased, or retrograde solubility.

That 86% of the randomly chosen proteins utilized in these studies demonstrated a temperature dependent solubility suggests that temperature induced crystallization could be a generally useful technique for production of protein crystals. Quantity, size and quality of crystals was influenced by position along the gradient of the block. Temperature has been shown to be a useful control parameter for obtaining not only solubility data but also preliminary crystallization data.

Acknowledgements This work was supported by a NASA contract (NAS8-97138). CrystalScoreTM was developed under a NASA Phase II SBIR grant (contract dNAS8-40820). We gratefully acknowledge the engineering support of Larry Kim and technical assistance of Vicki K. Johnson of the University of Alabama at Birmingham. We also wish to thank Gencor International, Inc. (Rochester, NY) for the gift of glucose isomerase.

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References [1] F. Rosenberger, E.J. Meehan, J. Crystal Growth 90 (1988) 74. [2] B. Lober, R. Giege´, J. Crystal Growth 122 (1992) 168. [3] R.C. DeMattei, R.S. Feigelson, J. Crystal Growth 122 (1992) 21. [4] R.C. DeMattei, R.S. Feigelson, J. Crystal Growth 128 (1993) 1225. [5] T.L. Bray, D.L. Powell, L.J. Kim, R.J. Gray, T. Le, R.P. Askew, M.D. Harrington, W.B. Rosenblum, W.W. Wilson, L.J. DeLucasm, AIAA 1995 Space Programs and Technologies Conf., 26—28, Huntsville, AL, 1995, p. 1. [6] R.J. Gray, L.J. Kim, L.J. DeLucas, A. Arabashi, W.W. Wilson, Conf. Proc. Space Technology Applications International Forum, Part II, 1997, p. 697. [7] R.J. Gray, J.B. Bishop, J.B. Kim, L.J. Bray, W.W. Wilson, L.J. DeLucas, A multichambered thermal gradient device

[8]

[9] [10] [11] [12] [13] [14] [15] [16]

(T-Block) for screening temperature induced protein crystallization, unpublished results. M.M. Long, J.B. Bishop, T.L. Nagabhushan, P. Reichert, G.D. Smith, L.J. DeLucas. J. Crystal Growth 168 (1996) 233. C.N. Pace, F. Vajdos, L. Fee, G. Grimsley, T. Gray, Protein Sci. 4 (1995) 2411. E.N. Baker, G. Dodson, J. Mol. Biol. 54 (1970) 605. W.J. Longley, Mol. Biol. 30 (1967) 323. W.B. Jakoby, Methods Enzymol. 11 (1971) 248. A.J. Hanson, Biol. Chem. 245 (1970) 4975. E. Cacioppo, M.L. Pusey, J. Crystal Growth 114 (1991) 286. F. Rosenberger, S.B. Howard, J.W. Sowers, T.A. Nyce, J. Crystal Growth 90 (1988) 74. G.D. Fasman (Ed.), Handbook of Biochemistry and Molecular Biology, Proteins, vol. II, 3rd ed., CRC Press, Cleveland, 1976.