The Crystallization of Acetylcholinesterase (AChE) - Defense

The Crystallization of Acetylcholinesterase (AChE) - Defense

AD 'ii1': W"CiYSTAL.IZATION OF A(.:tI.-IYLCI[OLINF.STErASE (ACHE) FROM TORPEDO EIEC'IRIC ORGAN ANNUM/FINAL REPORT I. SILMAN I.L. SUSSMAN July 18, 19...

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'ii1': W"CiYSTAL.IZATION OF A(.:tI.-IYLCI[OLINF.STErASE (ACHE) FROM TORPEDO EIEC'IRIC ORGAN

ANNUM/FINAL REPORT I. SILMAN I.L. SUSSMAN July 18, 1991 Supported by U.S. ARMY MEDICAL RESEARCH AND DEVELOPMUNiT COMMAND Fort Detrick, Frederick, Maryland 21702.5012

Contract No. DAMDI17-87-C-7003 Depts. of Neurobiology and Structural Chemistry Weiznann Institute of Sdience Rehovot 76100, Israel

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ApprovedI for public release; distribution unlimited. 'Ihe findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents.

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THE CRYSTALLIZATION OF ACETYLCHOLINESTERASE (AChE) FROM TORPEDO ELECTRIC ORGAN

ANNUAl/FINAL REPORT

I. SILMAN J.L. SUSSMAN

July 18, 1991

Supported by U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND Fort Detrick, Frederick, Maryland 21702-5012

Contract No. DAMDl7-87-C-7003

Depts. of Neurobiology and Structural Chemistry Weizmann Institute of Science Rehovot 76100, Isral

Approved for public release; distribution unlimited.

The findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents.

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It. TITLE (Include Security CI uslication) The Crystallization of Acetylcholinesterase I. PE.RSONA. AUTHOR(S)

Profs.

I.

Silman and J.L.

l3p. TYPE OF REPORT

From Torpedo Electric Organ

Sussman

13b. TIME COVERED 1 15 8 7

.1Annual-Final

(AChE)

FROM

/

/

OF REPORT (Year. Month, Day)

14. DAIT

TO

S/J4/.

IS. PAGE COUNT

7/18/91

I1. SUPPLEMENTARY NOTAT1ON Annual Il, FIELD

1988 -

15,

January

1989

14,

covers the period

of time January

COSATI CODES

I1. SUIJECT TERMS (Continue on reverse it necessary and identify by block number) RAVI Acetylcholinesterasel X-Ray CrystallographyI Torpedo Crystallization, Three-dimensional Structurel californical

GROUP

SUi-GROUP

01 06 Enzymest Acetylcholine is5 06 I1.ABSTRACT (Continue on reverse i necerury and Identify by block number)

of the enzyme acetylcholinesterase project is the crystallization The objective of this three-dimensional structure and, (AChE), with the long-term objective of determining its active site. thereby, the detailed topography of its organ was selected since It is a rich source of AChE and possesses an Torpedo electric enzyme A dimeric form of this amino acid sequence very similar to that of mammalian AChE. was purified by a procedure Ohich involved selective solubilization with a phosphatidylchromatography inositol-specific phospholipase C of bacterial origin, followed by affinity A highly purified ligand. employing a Sepharose conjugate of a suitable quaternary affinity AChE preparation was obtained in amounts which permitted a systematic attempt to crystallize the enzyme. In order to obtain a crystal form of the AChE preparation suitable for high-resolution we As a result conditions. crystallization X-ray studies, we examined hundreds of different ZI.ABSTRACT SECURITY CLASSIFICATION

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Virginia Miller

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19.

ABSTRACT (continued)

were able to obtain three (3) different crystal forms which diffract to One of these forms, which was obtained from better than 3 A resolution. ammonium sulfate-phosphate buffer, is capable of being ushock-cooled, to liquid nitrogen temperatures, which permitted X-ray data to be collected at This Oshock-cooling" procedure prolongs the lifetime of this temperature. the crystal in the X-ray beam almost indefinitely and has already permitted We are us to collect a preliminary set of three-dimensional X-ray data. thus in a position to proceed with the determination of the threedimensional structure of AChE.

Accesion For CRA&M TAB

NTIS DTIC

Urinnounced

Justification

................

By

Dist: ibution ! Availability Codes Dist

IT I

Avail andIor Special

I

3

D .CQUALITY rNMPE6M D

SUMMARY The objective of this project is the crystallization of acetylcholinesterase (AChE) from Torpedo electric organ, with the long-term goal of determining the three-dimensional structure of the enzyme and, thereby, the detailed topography of its catalytic site. Torpedo electric organ was selected since it is a rich source of AChE and possesses an amino acid sequence very similar to that of mammalian AChE. A dimeric form of this enzyme was purified by a procedure which involved selective solubilization with a phosphatidylinositol-specific phospholipase C

(PIPLC) of bacterial origin, followed by affinity chromatography employing a Sepharose conjugate of a suitable quaternary affinity ligand. A highly purified AChE preparation was obtained in amounts which permitted a systematic attempt to crystallize the enzyme. In order to obtain a crystal form of the AChE preparation suitable for high-resolution X-ray studies, we examined hundreds of different crystallization conditions. As a result we were able to obtain three different crystal forms, two of which diffract to better than 3 A resolution. One of these forms, which was obtained from ammonium sulfate in phosphate buffer, is capable of being "shock-cooled" to liquid nitrogen temperatures and permits X-ray data to be collected at this temperature. This "shock-cooling" procedure prolongs the lifetime of the crystal in the X-ray beam almost indefinitely and has already permitted us to collect a preliminary set of X-ray data up to 5 A resolution. We are thus in a position to proceed with the determination of the three-dimensional structure of AChE.

TABLE OF CONTENTS

Objectives

1

Background

2

Results and Discussion I. Purification of Acetylcholinesterase

3

II. Crystallization

3

Conclusions

6

List of Figures: Fig. 1: Schematic representation of the phosphatidylinositol-anchored dimer of AChE from Torpedo californica.

7

Fig. 2: Polyacrylamide gel electrophoresis in the presence of SDS of AChE from Torpedo electric organ purified by affinity chromatography subsequent to solubilization by PIPLC.

8

Fig. 3: Fractionation by high performance liquid chromatography (HPLC) of the Torpedo AChE dimer subsequent to purification by affinity chromatography.

9

Fig. 4: AChE crystals and X-ray diffraction from PEG 200

10

Fig. 5: AChE crystals and X-ray diffraction from 61% ammonium sulfate

11

Fig. 6: AChE crystals and X-ray diffraction from 57% ammonium sulfate

12

Table 1: AChE crystallization conditions

13

References

16

Distribution list

18

OBJECTIVES The objective of this project was the crystallization of acetylcholinesterase (ACHE) from Torpedo electric organ, with the long-term intention of determining the three-dimensional strucu=re of the enzyme and, thereby, the detailed topography of its catalytic site.

-I-

BACKGROUND In our original proposal we put forward a rationale for the choice of an AChE preparation suitable for attempts to grow crystals which could be used for the determination of the three-dimensional structure of this enzyme by Xray crystallography. We chose Torpedo electric organ as the source of the enzyme for three principal reasons: 1. Electric organ tissue of Torpedo californicacontains a high concentration of AChE and the tissue is readily available from commercial sources in kilogram quantities. Thus we considered it to be an excellent source for the amounts of highly purified AChE required for a project aimed at crystallization and eventual determination of the

three-dimensional structure of AChE. 2. The amino acid sequence of Torpedo AChE had been determined both by DNA cloning (1) and by direct sequencing (2) and the arrangement of its intrasubunit disulfide bonds had also been established (3). It appears to possess a high degree of homology, with respect to both sequence and disulfide bond arrangement, to all sequences of cholinesterases determined so far, i.e., human, bovine, mouse, mouse butyrylcholinesterase (BuChE), T. marmorata, Drosophila, human BuChE and rabbit BuChE as well as to rat carboxylesterase, rabbit carboxylesterase, and some other esterases (B.P. Doctor, private communication). Thus, information concerning its three-dimensional structure in general, and its active site in particular, should be highly pertinent to understanding the mode of action of the human enzyme and in devising therapeutic and prophylactic approaches to poisoning by organopbosphorus and carbamate nerve agents. 3. We have characterized a dimeric form of AChE from Torpedo electric organ which is bound to the plasma membrane via the diglyceride moiety of covalently attached phosphatidylinositol (PI) (Fig. 1) (5, 6). In this dimer the PI is attached to the COOH-terminus of each catalytic subunit through an intervening oligosaccharide sequence, which is apparently added post-translationally, and the diglyceride moiety of the PI serves as the hydrophobic anchor (7). The AChE dimer thus belongs to a recently described class of membrane proteins which are so attached to the plasma membrane (8). Such PI-anchored proteins can often be solubilized selectively by a Pl-specific phospholipase C of bacterial origin (8), and this is also the case for the Torpedo AChE dimer (9), thus allowing subsequent purification of the enzyme with minimum "lytic" damage or denaturation. For crystallographic studies, this preparation was expected to be preferable to preparations obtained by proteolytic digestion of the asymmetric forms of AChE from either Torpedo or Electrophorus,which yield an asymmetric tetramer which has undergone considerable proteolytic "nicking" (10). This is because "nicking" gives rise to a heterogeneity which may prove detrimental to crystal growth and may also hinder interpretation of the X-ray data if nicked and non-nicked forms coexist in the same crystal. As described below, we have been able, during the period covered by this Annual Final Report, to develop a procedure for reproducible and routine production of amounts of Torpedo electric organ AChE suitable for scanning a variety of crystallization conditions. We have, as a result, been able to obtain three crystal forms of the enzyme, two of which were found to diffract to a high resolution and have permitted us to collect preliminary crystallographic data. We are now in a position to proceed directly to studies leading to the solution of the threedimensional structure of this form of AChE.

-2-

RESULTS AND DISCUSSIONI I. Purificationof AChE Purification of AChE was carried out by an affinity chromatography procedure, subsequent to its solubilization by PIPLC from Staphylococcus aureus. The PIPLC, which is purified to homogeneity from the culture medium of the bacterium (11), was supplied by Dr. M.G. Low (Dept. of Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University). The particulate fraction from a homogenate of frozen electric organ tissue from Torpedo californica(obtained from Pacific Biomarine, Venice, CA) suspended in 0.1 M NaCI-0.01 M Tris, pH 8.0, was expoe,%d to PIPLC (1 pg/ml) for 18 hr at room temperature. As reported previously (9), almost quantitative solubilization of the G 2 AChE dimer was thus obtained. After high speed centrifugation at 78,000 g for 1 hr, at 40C, the supernatant was passed over an affinity chromatography column (bed volume ca. 7 ml), consisting of the affinity ligand (inaminophenyl)trimethylammonium, coupled to Sepharose 2B via a dicaproyl spacer (5, 12). After extensive washing, the bound AChE was eluted from the column with 2 mM decamethonium bromide in the application buffer. The purified enzyme was then dialyzed exhaustively against 0.1 M NaC1-0.01% sodium azide-1 mM 2[Nmorpholino]ethanesulfonic acid (NES), pH 6.5, and concentrated up to 10-15 mg/ml in a Centricon 30 microconcentrator (Amicon Co., Lexington, MA). The purified AChE displayed one major polypeptide band, of apparent molecular weight 65,000, on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of reducing agent. In the absence of reducing agent it migrated as a species of apparent molecular weight 130,000, corresponding to the disulfide-linked subunit dimer (Fig. 2). It displayed a catalytic activity of >2800 units per mg protein, using 3 mM [3H]acetylcholine as substrate, at pH 7.4 and 250 C. About 7 mg of purified AChE could be obtained routinely from ca. 450 g of electric organ tissue. An attempt to subject this affinity-purified AChE preparation to an additional purification step by high performance liquid chromatography (HPLC) did not improve the reproducibility of crystal production and/or the quality of the crystals obtained, even though a minor AChE component, which was consistently present in the PlPLC supernatant (Fig. 2) was separated from the main component by the HPLC procedure. 1I. Crystallization Attempts to crystallize Torpedo californica AChE were performed using the hanging drop method (14-16). This crystallization method utilizes the slow equilibration of concentration between a small drop containing the protein in dilute solution of the precipitating agent and a large volume reservoir containing the same precipitating agent at a higher concentration. Sealing of the protein drop and the reservoir from the outside atmosphere permits fine control of precipitation conditions. Screening of a wide variety of precipitation conditions, while using small quantities of protein, is made possible since the volume of each drop is 5-10 W1 and thus contains, at a final AChE concentration of ca. 5 mg/ml, a total of 25-50 pg protein. The screening of AChE crystallization conditions involved several stages. The first stage consisted of testing the most commonly used protein-precipitating agents. These include inorganic salts and various alcohols. The next stage involved the combination of various precipitating agents such as mixtures of different salts and of salts with various alcohols. Microcrystals of AChE were obtained under various crystallization conditions, but our first success in growing large single crystals of AChE of dimensions suitable for X-ray data collection, was achieved when using low molecular weight polyethylene glycol (PEG 200) as the precipitant. The crystals grew to 0.2-0.3 mnm in size, which is somewhat small unless the crystal diffraction is especially strong or the data are collected using a particularly strong X-ray source. -3-

Since the AChE crystals obtained from PEG 200 diffracted weakly, efforts were directed at obtaining larger ones. The technique used for increasing crystal size was that of seeding. In this technique a small or medium sized crystal, which has stopped growing spontaneously due to accumulation of imperfections or impurities on its surface, is transferred to a solution containing a slightly less concentrated precipitating agent. This solution is swirled gently around the crystal causing its outer surface to dissolve and thus exposing an uncontaminated fresh crystal surface. The seed crystal is then transferred to a fresh protein hanging drop where its clean faces can form an ideal nucleating environment for further crystal growth. This technique of seeding was used on AChE crystals growing in hanging drops containing 12 mg/mil protein 30% PEG 200 and 50 mM MES buffer, pH 6.0. Crystals 0.4 mm in size grew within 3 weeks from seed crystals which were 0.2 mm in size. The crystal cell parameters of this form were determined by X-ray measurements using a Rigaku AFCS-R rotating anode diffractometer operated at 10 kW. The crystals were found to be orthorhombic, space group P2221, with a = 163.4(.%0.2) A, b = 112.1(£-O.2) A and c = 81.3(O.1) X (Fig. 4). Assuming the unit cell to contain 4 AChE dimers, we calculated the ratio of the volume of the asymmetric unit to the molecular weight of the dimer (130,000) to be Vm = 2.95 A3/dalton, well within the range outlined by Matthews (17). The crystals diffract to 2.0 A resolution and lose ca. 25% of their difracting power within 24 hr in the X-ray beam at room temperature. An attempt to prevent the decay in crystalline order (which is caused by X-ray irradiation), by cooling the crystals to -150 0 C, resulted in total loss of diffracting power. Having found conditions for growing single crystals of AChE which diffract to high resolution, we embarked on exploring conditions to prolong the lifetime of the irradiated crystals in the X-ray beam. We sought, therefore, to grow a different crystal form which would be either less sensitive to X-ray irradiation or could survive the shockcooling technique. A method which has proved very successful for preserving the lifetime of a protein crystal under X-ray irradiation involves the shock-cooling of the crystal down to the temperature of boiling N2 (ca. -I 50 0C), followed by collection of diffraction data at this temperature. This method, which was developed for small molecule crystallography (19) and was extended in our laboratory to work with biological macromolecules (20), entails coating the crystal with a film of nonaqueous, viscous material such as oil, picking it up on a glass fiber by adhesion and transferring the fiber to the goniometer head in a position where a constant stream of boiling N2 gas is flowing over it. The advantages of this method are: 1) There is virtually no decay in the diffraction pattern after 2 weeks or more of X-ray irradiation. 2) The frozen crystal docs not slip during data collection. 3) Corrections to diffraction intensities due to absorption of X-rays by the glass capillary and the mother liquor, in which crystals are mounted for room temperature data collection, are minimized to only those corrections due to the crystal shape. 4) The thermal motion of atoms in the crystal is reduced, thus producing better data and easier interpretation of the electron density map. Use of the shock-cooling technique permits collection of an entire data set from just one crystal, thus avoiding errors arising out of the necessity of having to scale data sets from several crystals. Not every crystal can withstand shock-cooling without being shattered, and the experience accumulated in our lab shows that about 70% of the biological macromolecules survive it. Usually crystals grown from inorganic salt solutions survive it better than those grown from organic precipitants. As a consequence, we embarked on trying to grow AChE crystals from a -4-

wide combination of inorganic s.ats. Table 1 shows a list of crystallization conditions tested. After numerous a: .Aipts we were finally successful in crystallizing the AChE preparation from 61% (NH4) 2SO 4 in the presence of either 0.2 or 0.36 M phosphate buffer, pH 7.0. Crystals grew within 2 months to a size of 0.6 mm and diffracted to 2.5 A resolution (Fig. 5). The crystal cell parameters of this form were determined by X-ray diffr•,ction at -1500C using a Rigaku AFCS-R rotating anode diffractometer operated at 10 kW. The crystals were futnd to be trigonal, space group P3 121 or P3221, with a = b = l10.40(:tO.1) A, c = 136.95(±O.06)A, X--90W, •.,QJ, y1200 . Assuming the unit cell to contain 6 AChE dimers, we calculate the ratio of the volume of the asymmetric unit to the molecular weight of the monomer (65,000) to be Vm = 1.85 As/dalton. Sometimes, a third crystal form appeared from certain AChE preparations which precipitated under the conditions which yielded the trigonal form mentioned above (i.e., 61% (NH4)2SO 4, 0.36 M phosphate buffer, pH 7.0). These crystals appear to be orthorhombic (see Fig. 6) and grow to size of 0.5 mm in a few weeks from 57% (NH 4 )2SO 4 , 0.36 M phosphate buffer, pH 6.2. However, structurally, this crystal form seems to be much less mechanically robust than the trigonal form and is often damaged upon handling. A plausible explanation for this variability in crystal forms may arise from variability in the degree of glycosylation between different fish, a surface phenomenon likely to affect the inter-molecular packing forces, and hence the cryr= form, but not enzymatic activity. A similar occurrence was noted for the immature form of the enzyme, lacking a negatively charged sugar epitope, the situation being similar to that recently proposed for the G2 dimer in electric organ tissue of Torpedo marmorataby Bon et al. (13). Using the shock-cooling technique on the trigonal crystal form grown from ammonium sulfate, a preliminary diffraction data set of 2339 reflections was collected out to 5 A resolution at -15OPC. The two monitor reflections showed no decay in diffracting power over 21 hr of open shutter time (ca. 36 hr from the start of irradiation). Data were collected at a scanning motor rate of 4°/rain so as to ensure good statistics for the weak reflections. However, the quality of this trigonal crystal form is such that it will enable us to collect whole data sets up to 2.8A resolution from a single crystal, even at room temperature, without decay problems that are too severe.

-5-

CONCLUSIONS The work carried out during this project achieved its goals and justified the rationale which served as its basis. Thus the choice of the enzyme preparation, namely the dimeric form of AChE solubilized from electric organ tissue of Torpedo californica by PI-specific phospholipase C, has permitted us to conveniently obtain large amounts of intact, highly purified AChE. This, in turn, has led to successful crystallization experiments which have produced two crystal forms of the enzyme which diffract to high resolution. One trigonal crystal form, grown from ammonium sulfate-phosphate solutions, withstands shock-cooling to liquid nitrogen temperatures, and diffracts without irradiation decay at a temperature of-I 500C. The way is thus open to the solution of the three-dimensional structure of Torpedo AChE.

-6-

Model of the Membrane-Anchoring Domain of the Hydrophobic G 2 form of AChE from Torpedo californica NH2

ACHE

H

CATALYTIC SUBUNIT

S-s C-TERMINAL AMINO ACID ETHANOLAMINE

GLYCAN

NH2•• •

NHz

GLUCOSAMINE

INOSITOL

P

P

OUTER FACE MEMBRANE INNER FACE

Fig. 1: Schematic representation of the phosphatidylinositol-anchored dimer of AChE from Torpedo californica.

.7-

•, •

..

.--

LA

Fig. 2: Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of AChE from Torpedo electric organ purified by affinity chromatography subsequent to solubilization with PIPLC. Staining was with Coomassie Brilliant Blue. The consecutive lanes correspond to consecutive fractions obtained from the affinity column. The series on the left were electrophoresed in the absence of reducing agent and show the intact, disulfide-linked dimer. The series on the right shows the catalytic subunit monomer obtained in the presence of reducing agent. The constancy of the electrophoresis patterns across the elution profile suggests that the minor components observed, which are only seen on heavily overloaded gels, result from the presence of small amounts of "nicked" catalytic subunit polypeptides. -8-

I

|

I

I

Z

B

2000

A

""

._:I

>Z

1000 o

0- Cr 0-

20

25

30

35

20

25

30

35

FRACTION

FRACTION

Fig. 3: Fractionation by high performance liquid chromatography (HPLC) of the Torpedo AChE dimer subsequent to purification by affinity chromatography.

HPLC was performed on a Mono-Q anion exchange column (Pharmacia). The AChE was applied in 20 mM Tris chloride, pH 8.0 (ca. 10 mg of purified enzyme in 500 W. of buffer); it was eluted with a linear gradient of 0-0.8 M NaCI in the same buffer. Fractions of 0.5 ml were collected. The main peak of AChE was eluted at ca. 0.72 M NaCl and the small shoulder which preceded it was eluted at ca. 0.56 M NaCI. A) Elution profile obtained by continuous flow monitoring of protein absorption at 225 rnm. B) Elution profile obtained by monitoring AChE activity of individual fractions by the Ellmann procedure. -9-

(a)

(b,)

(C)

Fig. 4: AChE cryslas and X-ray diffraction from PEG 200.

(a) AChE crystas grown from PEG 200 (magnification X50). (b) Still X-ray diffraction from crysta grown from PEG 200.

(c) Precession X-ray diffraction from the same crystal.

-10-

(a)

(b)

(C)

Fig. 5: AChE crystals and X-ray diffraction from 61 %ammonium sulfate. (a)AChE crystals grown from ammonium sulfate (magnification X50). (b) Still X-ray diffraction from crystal grown from ammonium sulfate. (c) Precession X-ray diffraction from the same crystal.

-

11

-

I-I

Fig. 6: AChE crystals from 57% ammonium sulfate.

-12-

Table 1: AChE Crystallization Conditions

no.

drop

reservoir

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 22a 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

35% AS 40% AS 45% AS 30% AS 25% AS 20% AS 25% AS 50% AS 55% AS 45% AS 45% AS 45% AS 40% AS,0.086 M phosphate 40% AS,0.086 M phosphate 40% AS,0.086 M phosphate 45% AS,0.079 M phosphate 45% AS,0.079 M phosphate 27% AS,0.36 M phosphate 27% AS,0.36 M phosphate 40% AS,0.33 M phosphate 40% AS,0.1 M phosphate 27% AS,0.36 M phosphate 27% AS,0.36 M phosphate 24% MPD, 100 mM NaCI 21% MPD, 100 mM NaCl 16% MPD, 100 mM NaCl 13% MPD, 100 mM NaCI 10% MPD, 100 mM NaCI 7% MPD, 100 mM NaCl 25% PEG 200 25% PEG 200 25% PEG 200 15% PEG 200 20% PEG 400 14% PEG 400, 0.014M spermine 7% PEG 600 7% PEG 600 4% PEG 1000 4% PEG 1000 2% PEG 1500

45% AS 50% AS 55% AS 40% AS 35% AS 30% AS 35% AS 60% AS 65% AS 55% AS 55% AS 55% AS 50% AS 50% AS 50% AS 55% AS 55% AS 50% AS 60% AS 60% AS 60% AS 61% AS 57% AS 34% MPD 31% MPD 26% MPD 23% MPD 20% MPD 17% MPD 50% PEG 50% PEG 40% PEG 30% PEG 40% PEG 40% PEG 15% PEG 15% PEG 8% PEG 8% PEG 4% PEG

7.0 7.0 7.0 6.6 6.6 6.6 6.9 6.6 6.6 5.4 6.0 6.9 7.4 7.0 6.6 6.6 7.0 7.0 7.0 7.0 7.0 7.0 6.2 6.6 6.6 6.6 6.6 6.6 6.6 6.6 5.6 6.0 6.0 6.6 5.6 6.6 5.6 6.6 5.6 6.6

-13-

results -

-

large trigonal crystal large trigonal crystal large trigonal crystal 0.7 mm crystal, P3 121, LT 0.5 mm crystal, orthorhombic -

small crystal small crystal small crystal .38 mun crystal, P222 1, RT small plates small hexagons -

Table 1 (cont.)

no.

drop

reservoir

pH

results

40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79

10% PEG 1500 4% PEG 3350 2.86% PEG 3350 1.6% PEG 3350 5% PEG 3350,1 M NaCI 2% PEG 3350,1.5 M NaCI 2% PEG 3350,1.5 M NaCI 1.5% PEG 6000 1%PEG 6000 0.75% PEG 6000 5% PEG 6000,1 M NaCI 5% PEG 6000,1.5 M NaCI 5% PEG 6000,1.5 M NaCI 5% PEG 6000,1.5 M NaCI 2% PEG 6000,1.5 M NaCI 2% PEG 6000,1.5 M NaCI 2% PEG 6000,1.5 M NaCI 5% PEG 6000,0.5 M NaCI 2.5% PEG 8000 1.4% PEG 8000 4% PEG 8000 5% PEG 8000 3.3% PEG 8000 2.5% PEG 8000 2% PEG 8000 1.6% PEG 8000 5% PEG 8000,1 M NaCI 5% PEG 8000,1.5 M NaCI 5% PEG 8000,1.5 M NaCl 5% PEG 8000,1.5 M NaCI 2% PEG 8000,1.5 M NaCI 2% PEG 8000,1.5 M NaCI 2% PEG 8000,1.5 M NaCI 5% PEG 8000,0.5 M NaCI 4% PEG 20000 2.86% PEG 20000 2% PEG 20000 1.4% PEG 20000 0.5 M citrate 0.75 M citrate

20% PEG 6% PEG 5% PEG 3% PEG 10% PEG 5% PEG 5% PEG 3% PEG 2% PEG 2% PEG 10% PEG 10% PEG 10% PEG 10% PEG 5% PEG 5% PEG 5% PEG 10% PEG 15% PEG 10% PEG 8% PEG 10% PEG 6% PEG 5% PEG 4% PEG 3% PEG 10% PEG 10% PEG 10% PEG 10% PEG 5% PEG 5% PEG 5% PEG 10% PEG 6% PEG 5% PEG 4% PEG 3% PEG I M citrate I M citrate

6.6 6.6 6.6 6.6 6.6 5.4 6.0 6.6 .6.6 6.6 6.6 6.6 6.0 5.4 6.6 6.0 5.4 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.0 5.4 6.6 6.0 5.4 6.6 6.6 6.6 6.6 6.6 6.0 6.0

rods -

-

14-

-

-

-

Table I (cont.)

no.

drop

reservoir

pH

80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

2M phosphate 2M phosphate 2M phosphate 1.8 M phosphate 1.8 M phosphate 2M phosphate 1.8 M phosphate 30% ethanol 35% ethanol 25% ethanol 25% ethanol 25% ethanol 25% ethanol 25% ethanol 25% ethanol 25% ethanol 20% ethanol 10% ethanol 1 M Na2SO 4 1.3 M Na2SO 4 1.1 M Na2SO 4 1.1 M Na2SO 4 30% isopropanol 30% isopropanol 30% isopropanol 30% isopropanol

2.5 M phosphate 3 M phosphate 2.2M phosphate 2.2M phosphate 2.2M phosphate 2.2M phosphate 2.2M phosphate 50% ethanol 50% ethanol 50% ethanol 50% ethanol 50% ethanol 50% ethanol 50% ethanol 50% ethanol 50% ethanol 30% ethanol 20% ethanol 1.5 M Na2SO 4 2.6 M Na2SO 4 2M Na2SO 4 1.5 M Na2SO 4 50% isopropano 50% isopropano 50% isopropano 50% isopropano

7.0 7.0 6.6 6.6 7.0 7.4 7.4 7.0 7.0 7.8 7.4 7.0 6.6 6.2 5.8 5.4 7.0 7.0 7.0 7.0 7.0 7.0 6.6 7.2 7.4 7.8

AS = ammonium sulfate PEG = polyethylene glycol MPD = methylpentane diol RT = room temperature LT = low temperature - = no crystals

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results

poor crystals -

needles needles needles -

-

REFERENCES

(1) Primary Structure of Torpedo californica Acetylcholinesterase Deduced from its cDNA Sequence, (1986) Schumacher, M., Camp, S., Maulet, Y., Newton, M., MacPhee-Quigley, K., Taylor, S.S. and Taylor, P., Nature 319,407-409. (2) Primary Structure of the Catalytic Subunits from Two Molecular Forms of Acetylcholinesterase, (1985) MacPhee-Quigley, K., Taylor, P. and Taylor, S., J. Biol. Chem. 260, 12185-12189. (3) Profile of the Disulfide Bonds in Acetylcholinesterase, (1986) MacPhee-Quigley, K., Vedvick, T.S., Taylor, P. and Taylor, S.S., J. Biol. Chem. 261, 13565-13570. (4) Location of Disulfide Bonds within the Sequence of Human Serum Cholinesterase, (1987) Lockridge, 0., Adkins, S. and La Du, B.N., J. Biol. Chem. 262, 12945-12952. (5) Identification of Covalently Bound Inositol in the Hydrophobic Membrane-Anchoring Domain of Torpedo Acetylcholinesterase, (1985) Fuwrman, A.H., Low, M.G., Ackermann, K.E., Sherman, W.R. and Silman, I., Biochem. Biophys. Res. Commun. 129, 312-317. (6) Removal of Covalently Bound Inositol from Torpedo Acetylcholinesterase and Mammalian Alkaline Phosphatase by Nitrous Acid Deamination: Evidence for a Common Membrane-Anchoring Structure, (1987) Low, M.G., Futerman, A.H., Ackermann, K.E., Sherman, W.R. and Silman, I., Biochem. J. 241, 615-619. (7) Modes of Attachment of Acetylcholinesterase to the Surface Membrane, (1987) Silman, I. and Futerman, A.H., Eur. J. Biochem. 170, 11-22. (8) Covalently Attached Phosphatidylinositol as a Hydrophobic Anchor for Membrane Proteins, (1986) Low, M.G., Ferguson, M.A.J., Futerman, A.H.and Silman, I., Trends in Biochem. Sci. 11, 211-214. (9) Hydrophobic Dimer of Acetylcholinesterase from Torpedo californica Electric Organ is Solubilized by Phosphatidylinositol-Specific Phospholipase C, (1983) Futerman, A.H., Low, M.G. and Silman, I., Neurosci. Lett. 40, 85-89. (10) Molecular Structure of Elongated Forms of Electric Eel Acetylcholinesterase, (1978) Anglister, L. and Silman, I., J. Mol. Biol. 125, 293-311. (11) Phosphatidylinositol-Specific Phospholipase C from Staphylococcus aureus, (1981) Low, M.G., Methods Enzymol. 71, 741-746.

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(12) Major Component of Acetylcholinesterase in Torpedo Electroplax is not Basal Lamina Associated, (1980) Viratelle, OlM. and Bernhard, S.A., Biochemistry 19,4999-5007. (13) An Inmunoglobin M Monoclonal Antibody, Recognizing a Subset of Acetyicholinesterase Molecules from Electric Organs of Electrophorus and Torpedo, Belongs to the HNK-I Anticarbohydrate Family, (1987) Bon, S., Meflah, K., Musset, F., Grassi, J. and Massoulie, J., J. Neurochem. 49, 1720-1731. (14) Crystallization of Macromolecules: General Principles, (1985) McPherson, A., Methods Enzymol. 114, 112-120. (15) Use of Polyethylene Glycol in the Crystallization of Macromolecules, (1985) McPherson, A., Methods Enzymol. 114, 120-125. (16) Crystallization of Proteins by Variation of pH and Temperature, (1985) McPherson, A., Methods Enzymol. 114, 125-127. (17) Solvent Content of Protein Crystals, (1968) Matthews, B.W., J. Mol. Biol. 33, 491-497. (18) Purification and Crystallization of a Dimeric Form of Acetyicholinesterase from Torpedo californica Subsequent to Solubilization with Phosphatidylinositol-Specific Phospholipase C, (1988) Sussman, J.L., Harel, M., Frolow, F., Varon, L., Toker, L., Futerman, A.H. and Silman, I., J. Mol. Biol. 203, 821-823. (19) Cryocrystallography of Biological Macromolecules: A Generally Applicable Method, (1988) Hope, H., Acta Cryst. B44, 22-26. (20) Macromolecular X-Ray Data Collection on a Rotating Anode Difffractometer, (1987) Hope, H., Frolow, F. and Sussman, iLL., Rigaku J. 4, 3-10.

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DAMD17-87-C-7003 List of Publications 1.

J.L. Sussman, M. Harel, F. Frolow, L. Varon, L. Toker, A. H. Futerman & I. Silman (1988). "Purification and Crystallization of a Dimeric Form of Acetylcholinesterase from Torpedo californica Subsequent to Solubilization with Phosphatidylinositol-Specific Phospholipase C". J. Mol. Biol. 203, 821-823.

2.

J.L. Sussman, M. Harel, F. Frolow & 1. Silman (1989). "X-ray crystallographic studies of acetylchotinesterase" in 1989 U.S. Army Proc. Medical Defense Bioscience Review, Columbia, MD, 309-316

List of Personnel Receiving Contract Support 1. 2. 3. 4. 5. 6.

Prof. Israel Silman Prof. Joel L. Sussman Dr. Lilly Toker Dr. Nitzah Steinberg Dr. Yaron Levine Mr. Yaacov Shabtai

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