- Email: [email protected]
Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants
Journal of Crystal Growth 122 (1992) 385—392 North-Holland
o~o~
CRYSTAL GROWTH
Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants G.E. Schulz ~, M. Dreyer, C. Klein, A. Kreusch, P. Mitt!, C.W. Muller, J. Müller-Dieckmann, Y.A. Muller, K. Proba, G. Schlauderer, P. Spürgin, T. Stehie and M.S. Weiss Inst itut für Organische Chemie und Biochemie der Unitersithi, Albertstrasse 21, D-W-7800 Freiburg i. Br., Germany
Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8 A resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0 A resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.
1. Introduction The major concern of our laboratory is the structure elucidation of proteins by X-ray diffraction and amino acid sequence analysis. In addition, the established recombinant gene technology has been adopted to produce large quantities of native and mutated proteins in bacteria. Of particular interest is the combination of structural and functional studies. Since our structure analyses are based on X-ray diffraction, we are bound to produce X-ray grade crystals of all proteins of interest. Experiments over the years showed that protein preparation and stabilization are the most important aspects for obtaining good crystals. In our studies no case turned out where crystallization depended strongly on the applied technique. Therefore, we generally apply the convenient hanging drop method with Linbro plates and To whom correspondence should be addressed, 0022-0248/92/$05.00 © 1992
—
siliconized cover slides. Depression s!ides, batch crystallization of sitting-drop apparatus are rarely used. Also, changes in the physical environment as the application of electric or magnetic fields or high (centrifuge) and very low (space experiments) gravitational forces are not applied. Participation in one microgravity experiment [1] convinced us that gravitation is of minor importance at best. Given a pure protein, the important parameters for crystallization are the pH, precipitant, temperature, detergents (for membrane proteins) and additives like divalent cations, substrates, inhibitors and organic solvents. In general these parameters are screened extensively. In the following, we present the crystals that are currently analyzed in our laboratory. In order to keep our research coherent, only a limited number of protein families are tackled: (i), channd-forming integral membrane proteins, (ii), nucleoside-monophosphate kinases, (iii), FAD-containing oxidoreductases, and (iv), sugar-processing enzymes.
Elsevier Science Publishers B.V. All rights reserved
386
G.E. Schulz et a!. / H,ghl~ordered crystaLs of channel-forming membrane proteins
C
Fig. 1. Protein crystals produced with the hanging drop method the obtained maximum sizes are stated in the text: (a) crystal of the channel-forming membrane protein porin from Rh. capsulatus; (b) crystal of the channel-forming membrane protein porin from Rh. blastica; (b) crystal of the major variant AK2 1 of the adenylate kinase of the mitochondrial intermembrane space, which was obtained after removing the minor variant AK22 by chromatofocussing; (d) crystal of guanylate kinase; (e) crystal of cyclodextrin glycosyltransferase of B. circulans.
G.E. Schulz et al.
/ Highly ordered crystals of channel-forming membrane proteins
2. Crystallization of integral membrane proteins
387
Table 1 Abbreviations
Among the large number of membrane proteins, we are concerned with porins. Porins are located in the outer membrane of Gram-negative bacteria, mitochondria and chloroplasts. They usually form trimers that are very stable towards SDS, heat and proteases. The M r of porins range between 30,000 and 50,000 per subunit. X-ray grade porin crystals have been available for some time [2], but only recently has a porin structure been solved [3]. Porins form aqueous channels which allow for the diffusion of small hydrophilic molecules with exclusion limits typically around 600 Da. Some porins act as phage receptors and some are major antigens and exhibit pathogenic properties. Structurally, porins represent a new class of proteins, as they consist of a 16-stranded a-barrel. In contrast, the majority of membrane proteins is most likely a-helical. 2.1. Porin from Rhodobacter capsulatus Crystals of this porin have been first produced by Nestel et al. [41. They diffracted to about 3 A resolution. The structure of these crystals has been solved by multiple isomorphous replacement [5,6]. Since the crystallization failed for some period of time, the preparation procedure was revisited and modified. As a result, a new crystal form was found that diffracts to 1.8 A resolution and yields crystals routinely [7]. The major changes were the removal of SDS and EDTA from the preparation. Presumably, SDS had caused conformational heterogeneity and EDTA had removed the structurally important calcium ions [3]. After a peptide-based sequence analysis [81,the structure of this porin was solved at 1.8 A resolution by molecular replacement [3], yielding the coordinates of all 301 amino acid residues, 3 Ca2~ ions, 4 detergent molecules and 274 water molecules per subunit. See table 1 for abbreviations. The high resolution crystal form was obtained at 20°C,using the hanging drop method with 10 p.! drops containing 5 mg/ml protein in Tris at pH 7.2, 0.6% n-octyltetraoxyethylene, 0.3M LiC1 and 9% PEG-600. The reservoir solution was the
AK1, AK2, AK3 AKeco AKyst DTT Ap~A EDTA MES MOPS
Isoenzymes of mammalian adenylate kinases Adenylate kinase from E. co/i Adenylate kinase from yeast 1, P5-bis(adenosine-5’-)pentaphosphate PDithiothreitol Ethylenediaminetetraacetate Morpholinoethanesulfonate Morpholinopropanesulfonate
PEG SOS TPP Tris
Polyethyleneglycol Sodium dodecylsulfate Thiaminepyrophosphate Tris(hydroxymethyl)aminomethane
same but without porin and with 27% PEG-600. The space group is R3 (a b 92.3 A, c 146.2 A) with 1 subunit per asymmetric unit. As shown in fig. la, the crystal habit is rhombohedral with edge lengths of up to 600 p.m. =
=
=
2.2. Porin from Rhodopseudomonas blastica This porin is another member of the family of pore forming proteins in the outer membrane of Gram-negative bacteria. Like most other porins it forms trimers. Preliminary sequence data show only low homology (around 25% identical amino acids) with the porin from Rhodobacter capsulatus. For crystallization, we used the hanging drop method at 20°C. The drop contained 5 mg/ml protein, 20mM Tris at pH 6.8, 0.35M LiCl, 0.6% n-octyltetraoxyethylene and 17% PEG-600. The reservoir was at 35% PEG-600. Surprisingly, these conditions are almost the same as for the porin from Rh. capsulatus. The crystals grow routinely within about 2 weeks to a maximum size of 400 X 400 x 350 p.m3. The best crystals are nearly cubic in shape, as shown in fig. lb. The space group is R3 (a b 104.3 A, c 125.1 A) with 1 subunit per asymmetric unit. They diffract to at least 2.0 A. Native X-ray diffraction data to 2.3 A resolution have been collected on a Xentronics area detector installed on a Rigaku rotating anode tube. The structure analysis is in progress. =
=
=
388
G.E. Schulz et a!.
/ Highly ordered crystals
3. Crystallization of nucleoside-monophosphate kinases
of channel-forming membraneproteins
maximum size of 1000 X 800 X 600 p.m3. X-ray diffraction data were collected to 2.1 A resolution. In the case of double mutant Asp89 Val/ Arg165 —s Ile, the crystals grew within a few days to sizes around 500 x 300 X 100 p.m3. The most —*
These enzymes transfer a phosphoryl group onto a phosphoric acid forming an anhydride bond according to the reaction: Mg2~N 1TP + 2~+N N2MP N~DP+ Mg 2DP, where N1 and N2 are 2 nucleotide bases. Since this transfer is energetically much less favorable than the transfer to water forming orthophosphate, these kinases have to take great care to exclude water from their active center in order to avoid undesirable NTP hydrolysis, i.e. destruction of free energy. Given these conditions, the enzymes are remarkably small with Mr in the range 20,000 to 25,000. As a consequence, one has to expect large induced-fit movements during catalysis, which indeed have been observed [91. This obstructs soaking experiments with substrates as these are bound to break the crystals. Thus, all structural data on the catalytic mechanism have to be derived from cocrystais with ligands. We have analyzed a number of native and mutated enzymes of this group in free and in ligated forms. For each analysis, a new crystallization procedure had to be established. 3.1. Mutants of adenylate kinase from yeast ligated with substrates The structure of this enzyme has been solved some time ago [10]. Moreover, the AKyst gene has been cloned into E. coli to facilitate the preparation of large amounts of the protein and to carry out site-directed mutagenesis. A catalytic inactive double mutant (Asp89 —s Val, Arg165 —s lie) has been cocrystallized with ATP and AMP. Mutant 1le213 —s Phe has an exchange in the hydrophobic core decreasing its thermostability. This mutant has been crystallized with the twosubstrates-mimicking inhibitor Ap5A under essentially same,out, conditions the wildtype enzyme. It the turned however,as that this mutant
regularly shaped crystals are obtained by macroseeding and by the addition of KCI and f3-octylglucoside. Data were collected to 2.7 A resolution. Double mutant Asp89 2Val/Arg165 lIe 2~2 crystallized in space group P 1 1(a 48.2 A, b 73.4 A, c 119.8 A) with 2 molecules per asymmetric unit. The drop contained 50mM imidazole at pH 6.2, 21% PEG-3350, 2.5mM ATP and AMP, 10 mg/mI protein, 0.05% /3-octylglucoside and 75mM KC1. The reservoir was 50mM imidazole at pH 6.2 with 28% PEG-3350. In contrast, the wildtype enzyme and mutant 1le213 -s Phe crystallize in space group P1 (a 36.0 A, b 40.4 A, c 45.5 A, a 110.7°, /3 108.80, y 64.7°)with I molecule per asymmetric unit. In these cases the drop contained 60mM imidazole at pH 7.2, 26% PEG-8000, 2mM Ap5A, 12 mg/mi protein and (only for the mutant) 2mM /3-mercaptoethanol. The reservoir was 60mM imidazole at pH 7.2 with 29% PEG-8000. —,
—‘
=
=
=
=
=
=
=
=
=
3.2. Cocrystals of wildtype and mutant adenylate kinase from E. coli and inhibitor The structure of AKeco in complex with the two-substrate-mimicking inhibitor Ap5A has been solved [11]. The final R-factor is 19.6% at a resolution of 1.9 A. The structures of two mutants of AKeco were determined at 2.4 and 3.4 A resolution, respectively. For the crystallization of the wildtype enzyme, we used the hanging drop method at 20°C.The drop contained 22 mg protein/mI, 2mM Ap5A, 50mM MES at pH 6.7, 1.5% PEG-1500 and 1.5M ammonium sulfate. The reservoir had 50mM MES at pH 6.7 with 2.1M ammonium sulfate. Crystals grew within 3.4 days space to a maximum The group is size P2 of 1200 x 500 x 150 p.m 1221 (a 73.2 A, b 79.8 A, c 84.8 A) with 2 molecules per asymmetric unit. A substantial improvement with respect to size and reproducibility of the crystals was achieved by addition of PEG-1500, which re=
required /3-mercaptoethanoi for crystal growth. Crystallization was carried out at 20°C using the hanging drop method. With mutant 1le213 —s Phe, the crystals grew within a few days to a
=
=
G.E. Schulz eta!.
/ Highly ordered crystals of channel-forming membrane proteins
389
duced the tendency of the crystals to grow attached to each other. Mutant GlylO —s Va! crystallizes under wild-
unit. The crystals diffract to at least 1.9 A resolution. The structure has been solved and is currently being refined. The minor component AK22
type conditions in the same space group, but with a c-axis prolonged by 1.5%. The mutant crystals tend to grow bigger in size than the wildtype crystals. Mutant Pro9 —s Leu yielded only thin crystal plates under wildtype crystallization conditions. By addition of 2% ethanol, we could obtain a new crystal form that diffracts reasonably well. The space group is P21 (a = 60.3 A, b 77.8 A, c 57.7 A, /3 = 94.3°)with 2 molecules per asymmetric unit. Wildtype AKeco and mutant Pro9 Leu were also crystallized under microgravity conditions. Both proteins were sent into space using an unmanned Chinese re-entry system and were incubated there for 8 days (COSIMA-I). This experiment yielded crystals of the wildtype and the mutant enzyme. Size and quality of these crystals, however, did not reach the quality of the crystals grown in the home laboratory [1].
crystallized under similar conditions. The obtamed crystals, however, are not yet suitable for X-ray analysis.
=
=
—,
3.3. Mitochondrial adenylate kinase In mammalian cells there are three adenylate kinase isozymes: AK1 in the cytosol, AK3 in the mitochondrial matrix and AK2 in the mitochondna! intermembrane the structures of AK1 [12] and AK3space. [131 While have been solved at high resolution, AK2 has resisted crystallization for many years. Recently, we discovered that the enzyme preparation consists of 2 variants, AK2 1 and AK22, that differ in 3 amino acids from each other. After separating these species on large scale by chromatofocussing, we succeeded to grow crystals from both of them using the hanging drop method at 20°C.After macroseeding, the major component AK21 grew within 5 days 3. to A maximum crystal is sizes around 1500 500 drop x 180contained p.m depicted in fig. ic. XThe 7 mg/ml enzyme, 20mM MOPS at pH 7.1, 5mM /3mercaptoethanol, 5mM MgSO 4 and 9% PEG3350. The reservoir was 20% PEG-3350, 5mM f3-mercaptoethanol in 20mM MOPS at pH 7.1. The space group is P2~2~21 (a = 44.3 A, b = 50.1 A, c = 122.3 A) with 1 molecule per asymmetric
3.4. Guanylate kinase ligated with GMP Guanylate kinase resembles the adenylate kinases with respect to size and reaction mechanism. The enzyme from yeast has been crystallized in complex with its substrate GMP and the structure has been determined at 2.0 A resolution [14]. Two crystal forms were obtained. Crystal form I belongs to space group P432~2(a b 50.8 A, c 155.3 A) with 1 molecule per asymmetric unit. Crystal form II has space group C2221 (a = 71.2 A, b = 72.3 A,. c = 152.7 A) with 2 molecules per asymmetric unit. Both crystals forms grow at the same conditions and can be interconverted by temperature changes: form I predominates at 4°Cwhereas form I and form II occur equally distributed at 25°C, even in one drop. The interconversion corresponds to small changes in the molecular packing. Using the hanging drop method, crystals grew within about 3 days 3,upastoshown a maximum sizeThe of in fig. ld. 1200 X 600 X 400 p.m crystal size increased dramatically when PEG1000 was added until phase separation occurred. The drop contained 7 mg/ml enzyme, l.OM am=
=
=
monium sulfate, 1mM GMP, 1% PEG-bOO, 40mM potassium phosphate at pH 5.5. The reservoir was 2.OM ammonium sulfate, 40mM potassium phosphate at pH 5.5. During the structure analysis all crystal handling had to be done in the mother liquor, since no enzyme-free stabilizing solution could be found. 3.5. Uridylate kinase Uridylate kinase from yeast is homologous (40% sequence identity) to AK1. It was crystallized using the hanging drop method with PEG as precipitant. The addition of up to 0.025% /3-octylglucoside prevented the formation of microcrystalline material and increased the crystal size
390
G.E. Schulz et a!.
/ Highly ordered crystals of channel-forming membrane proteins
up to a maximum size of 700 X 300 x 300 p.m3. The space group is P6 1522 (a = b 64.2 A, c = 185.0 A) with one molecule per asymmetric unit. The crystals diffract to at least 2.1 A resolution. The structure analysis is under way. =
4. Crystallization of flavo-enzymes In addition to human glutathione reductase [15] which has now become a model enzyme for the increasing family of FAD-containing oxidoreductases, several other flavo-enzymes are now investigated in our laboratory. We are particularly interested in the reaction mechanisms and evolutionary relationships of these enzymes. Recently a scientifically and technically important mutant of glutathione reductase that changed the cofactor specificity from NADP to NAD [16] attracted widespread attention. We are currently analyzing this change in order to explain it structurally. 4.1. Wildtype and mutant glutathione reductase from E. coli The enzyme reduces glutathione at the expense of NADPH. It consists of 2 identical subunits with Mr = 48,700 each. Its structure is known at 3 A resolution [17]. Using the hanging drop method, the enzyme was crystallized in 3 different forms, 2 of which were suitable for X-ray analysis.crystals grow in drops containing Form-PlO 5% PEG-10000 (Fluka), 20mM potassium phosphate at pH 5.4 and 15 mg/rn! protein. The reservoir had 20% PEG-10000, 20mM potassium phosphate at pH 5.4. The maximum crystal size was 2500 x 800 x 800 p.m3. The space group is P4 3212 (a = b = 62.0 A, c = 336.5 A) with 1 subunit per asymmetric unit. The crystals diffract anisotropicall~’to 3.0 A resolution along the c-axis and to 3.4 A resolution along a and b. The anisotropic diffraction of form-P 10 seems to be explained by the few observed contacts in the a—b plane. Therefore, we attempted to improve the packing order by site-directed mutagenesis
inserting a new contact in this plane. The crystallization of this mutant enzyme is in progress. Form-P8 crystals grow in drops of 7% PEG8000 (Sigma), 100mM potassium phosphate at pH 5.5, 20 mg/mI protein using a reservoir of 20% PEG-800 and 100mM potassium phosphate at pH 5.5. After macnoseeding with 14% crystals size PEG50 X 3 (washed with andof7% 8000 solutions), we obtained crystals with a maxi50 X 50 p.m mum size of 800 X 400 X 300 p.m3. The space group is P2
1 (a = 120.5 A, b = 73.5 A, c = 60.5 A, yThe = 82.7°)with 2 subunits per asymmetric unit. crystals diffract to 2.0 A resolution. Form-P8 is very difficult to reproduce without seed crystals. Only about 1% of the crystallization attempts were successful. 4.2. NADH peroxidase NADH peroxidase is a tetrameric flavo-enzyme (symmetry D2) with an Mr of 50,300 per subunit. The enzyme catalyzes the degradation of H202 at the expense of NADH, FAD and an unusual stabilized cysteine-sulfenic acid participate in catalysis. Normally, H2O2 reduction is catalyzed by heme- or selenium-containing proteins. NADH peroxidase crystallizes in the space group 1222 (a = 77.2 A, b = 134.5 A, c = 145.9 A). The crystals diffract to 2.1 A resolution and contain 1 subunit per asymmetric unit. The structure has been determined [18]. Using the hanging drop method, crystals grew within 3 days up toofa 3. Most maximum size of 1000 x 500 X 300 p.m them were of flowerlike shape. The drop contamed 5 mg/rn! enzyme, 1.05M ammonium sulfate, 2mM DTT, 2.5 p.M FAD, and 50mM potassiurn phosphate at pH 7.0. The reservoir was 2.1M ammonium sulfate, 2mM DTT, 5p.M FAD and 50mM potassium phosphate at pH 7.0. 4.3. Pyruvate oxidase The enzyme from Lactobacillus plantarum is a homotetramer with an Mr of 263,000. It contains the coenzymes FAD and TPP. For crystallization we applied the hanging drop method. The enzyme crystallizes readily in form A using 20mM
G.E. Schulz et al.
/ Highly ordered crystals of channel-forming membrane proteins
phosphate at pH 5.7—6.5, 20 mg/ml protein, O.5M ammonium sulfate in the drop, and the same buffer with 1M ammonium sulfate in the reservoir. The average crystal size was 700 x 700 x 300 p.m3. Because of its extremely high mosaicity, crystal form A is not suitable for X-ray analysis. After a tedious search, we could eventually produce crystals of form B using drops with 20mM phosphate at pH 5.2, 1mM TPP, 20 mg/ml protein, O.5M ammonium sulfate and 5% glycerol. The reservoir contained the same buffer and 1.OM ammonium sulfate with 10% glycerol. Crystals grow within 6 months to a maximum size of 1000 x 90 x 90 p.m3 in a small percentage of the drops. The crystals are well ordered and diffract to beyond 2.4 A resolution. The space group is C222 1 (a = 121.3 A, b = 154.7 A, c = 166.7 A) with analysis 2 subunits asymmetric unit. The structure is inper progress.
5. Crystallization of sugar-processing enzymes This group of enzymes is most important for the food and drug industry. One may expect future applications in other parts of the chemical industry because saccharides are among the cheapest organic raw materials. We are analyzing cyclodextrin glycosyltransferase, which catalyzes the conversion of starch to cyclodextrins, widely used as micro-encapsulators. Furthermore, we work on an aldolase which may become important for the production of defined chiral cornpounds as building blocks for organic synthesis [19]. Here, we intend to change substrate specificities by site-directed mutations. 5.1. Cyclodextrin glycosyltransferase The enzyme from Bacillus circulans is monomeric and consists of 684 amino acid residues. The structure of the wildtype enzyme was refined at 2.0 A resolution with an R-factor of 17.6% [20]. Nine mutants (Pro138 —s Ala, Tyrl9l —s Phe, Tyr195 Phe, Asp229 —s Ala, Phe259 —s Leu, Ser276 —s Cys, Asp328 —s Ala, Met329 —s Asn and Ser428 —s Cys) were crystallized isomorphously with the wildtype enzyme. ~
—
391
The Cys-mutants yielded useful mercury derivatives that improved the multiple isomorphous replacement electron density map. The structures of mutants Pro138 —s Ala, Asp229 Ala, Phe259 Leu, Asp328 Ala and Met329 —s Asn were refined at 2.5 and 2.7 A resolution. The wildtype enzyme crystallizes in batches over time spans of 3 to 12 months. For the mutant enzymes we used hanging drops at similar conditions as with the wildtype, but macroseeding was done with wildtype crystals. The drops contamed 0.6M ammonium sulfate, 50mM tnethanolamine, 50mM citric acid at pH 6.7, 0.5% PEG-1500 and 11 mg/mI protein. The reservoir was 0.9M ammonium sulfate, 50mM tnethanolamine, 50mM citric acid at pH 6.7 and —
—‘
—
0.5% PEG-l500. The obtained 3maximum crystal for the mutants, sizes 3000 werex1500 100 for p.mthe wildtype. One and 300 xx 150 300 xp.m3 example is shown in fig. le. The crystal space group is P2 12121 (a =94.8 A, b=104.7 A, c= 114.0 A) with 1 molecule per asymmetric unit. The diffraction pattern extends to 1.9 A resolution. 5.2. Fuculose-1-phosphate aldolase enzyme from E. coli and belongs to Zn the2~for class II The (metal binding) aldolases needs catalytic activity. The native enzyme is a homotetramer with an Mr of 24,000 per subunit. It is prepared from an overexpressing strain by a three-step purification consisting of ion exchange, hydrophobic interaction and gelpermeation chromatography. For crystallization we used the hanging drop method at 20°Cand ammonium sulfate as a precipitant. Within about 10 days crystals with a maximum size of 500 x 500 x 110 p.m3 were obtained. They belong to space group P42 12 and diffract to a resolution of 2.2 A. They contain one subunit per asymmetric unit. The structure analy. sis is in progress. 6. Conclusions Today the crystallization problem is obviously different from a decade ago, the most drastic
392
G.E. Schulz el a!.
/ Highly ordered crystals of channel-forming membrane proteins
change being the availability of large amounts of recombinant proteins. Having enough material, a large number of crystallization parameters can be screened conveniently. But even more important is the dramatic shortening of the isolation procedures for recombinant proteins. While purification factors of the order of a million are required to isolate a protein from natural living material, this factor is reduced to the order of a hundred in reasonably well-expressed recombinant proteins. Cutting the isolation procedure short means reducing the strain on the protein, which leads to conformationally more homogeneous proteins that crystallize more readily. We also find that conformational homogeneity can often be enhanced by cocrystallization with protein ligands. Taken together, one may expect that in the future crystallization will become less and less of an obstructing road block in structure analyses.
References [I] V.A. Erdmann, C. Lippmann, C. Betzel, Z. Dauter, K. Wilson, R. Hilgenfeld, J. Hoven, A. Liesum, W. Saenger. A. Miiller-Fahrnow, W. Hinrichs, M. DOvel, G.E. Schulz, C.W. MOller, HG. Wittmann, A. Yonath, G. Weber, K. Stegen and A. Plaas-Link, FEBS Letters 259 (1989) 194. [2] R.M. Garavito and J. Rosenbusch, J. Cell Biol. 86 (1980) 327.
[3] M.S. Weiss, A. Kreusch, E. Schiltz, U. Nestel, W. Welte, J. Weckesser and G.E. Schulz, FEBS Letters 280 (1991) 379. ~ U. Nestel, T. Wacker, D. Woitzik, J. Weckesser, W. Kreutz and W. Welte, FEBS Letters 242 (1989) 405. [5] MS. Weiss, U. Nestel, T. Wacker, J. Weckesser, W. Kreutz and G.E. Schulz, FEBS Letters 256 (1989) 143. [6] M.S. Weiss, T. Wacker, J. Weckesser, W. Welte and G.E. Schulz, FEBS Letters 267 (1990) 268. [7] A. Kreusch, MS. Weiss, W. Welte, J. Weckesser and G.E. Schulz, J. Mol. Biol. 217 (1991) 9. [8] E. Schiltz, A. Kreusch, U. Nestel and G.E. Schulz, European J. Biochem. 199 (1991) 587. [9] G.E. Schulz, C.W. Muller and K. Diederichs, J. Mol. Biol. 213 (1990) 627. [10] U. Egner, AG. Tomasselli and G.E. Schulz, J. Mol. Biol. 195 (1987) 649. [11] C.W. Muller and G.E. Schulz, J. Mol. Biol. 202 (1988) 909. [12] D. Biol.Dreusicke, 199 (1988) PA. 359. Karplus and G.E. Schulz, J. Mol. [13] K. Diederichs and G.E. Schulz, J. Mol. Biol. 217 (1991) 541. [14] T. Stehle and G.E. Schulz, J. Mol. Biol. 211 (1990) 249. [15] PA. Karplus and G.E. Schulz, J. Mol. Biol. 210 (1989) 163. [16] N.S. Scrutton, A. Berry and R.N. Perham, Nature (London) 343 (1990) 38. [17] U. Ermler and G.E. Schulz, Proteins: Struct. Funct. Genet. 9 (1991) 174. [181 T. Stehle, S.A. Ahmed, A. Claiborne and G.E. Schulz FEBS Letters 267 (1990) 186. [19] W.-D. F~ssner,G. Sinerius, A. Schneider, M. Dreyer, G.E. Schulz, J. Badia and J. Aguilar, Angew. Chem. Intern. Ed. EngI. 30 (1991) 555. [20] C. Klein and G.E. Schulz, J. Mol. Biol. 217 (1991) 737.
o~o~
CRYSTAL GROWTH
Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants G.E. Schulz ~, M. Dreyer, C. Klein, A. Kreusch, P. Mitt!, C.W. Muller, J. Müller-Dieckmann, Y.A. Muller, K. Proba, G. Schlauderer, P. Spürgin, T. Stehie and M.S. Weiss Inst itut für Organische Chemie und Biochemie der Unitersithi, Albertstrasse 21, D-W-7800 Freiburg i. Br., Germany
Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8 A resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0 A resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.
1. Introduction The major concern of our laboratory is the structure elucidation of proteins by X-ray diffraction and amino acid sequence analysis. In addition, the established recombinant gene technology has been adopted to produce large quantities of native and mutated proteins in bacteria. Of particular interest is the combination of structural and functional studies. Since our structure analyses are based on X-ray diffraction, we are bound to produce X-ray grade crystals of all proteins of interest. Experiments over the years showed that protein preparation and stabilization are the most important aspects for obtaining good crystals. In our studies no case turned out where crystallization depended strongly on the applied technique. Therefore, we generally apply the convenient hanging drop method with Linbro plates and To whom correspondence should be addressed, 0022-0248/92/$05.00 © 1992
—
siliconized cover slides. Depression s!ides, batch crystallization of sitting-drop apparatus are rarely used. Also, changes in the physical environment as the application of electric or magnetic fields or high (centrifuge) and very low (space experiments) gravitational forces are not applied. Participation in one microgravity experiment [1] convinced us that gravitation is of minor importance at best. Given a pure protein, the important parameters for crystallization are the pH, precipitant, temperature, detergents (for membrane proteins) and additives like divalent cations, substrates, inhibitors and organic solvents. In general these parameters are screened extensively. In the following, we present the crystals that are currently analyzed in our laboratory. In order to keep our research coherent, only a limited number of protein families are tackled: (i), channd-forming integral membrane proteins, (ii), nucleoside-monophosphate kinases, (iii), FAD-containing oxidoreductases, and (iv), sugar-processing enzymes.
Elsevier Science Publishers B.V. All rights reserved
386
G.E. Schulz et a!. / H,ghl~ordered crystaLs of channel-forming membrane proteins
C
Fig. 1. Protein crystals produced with the hanging drop method the obtained maximum sizes are stated in the text: (a) crystal of the channel-forming membrane protein porin from Rh. capsulatus; (b) crystal of the channel-forming membrane protein porin from Rh. blastica; (b) crystal of the major variant AK2 1 of the adenylate kinase of the mitochondrial intermembrane space, which was obtained after removing the minor variant AK22 by chromatofocussing; (d) crystal of guanylate kinase; (e) crystal of cyclodextrin glycosyltransferase of B. circulans.
G.E. Schulz et al.
/ Highly ordered crystals of channel-forming membrane proteins
2. Crystallization of integral membrane proteins
387
Table 1 Abbreviations
Among the large number of membrane proteins, we are concerned with porins. Porins are located in the outer membrane of Gram-negative bacteria, mitochondria and chloroplasts. They usually form trimers that are very stable towards SDS, heat and proteases. The M r of porins range between 30,000 and 50,000 per subunit. X-ray grade porin crystals have been available for some time [2], but only recently has a porin structure been solved [3]. Porins form aqueous channels which allow for the diffusion of small hydrophilic molecules with exclusion limits typically around 600 Da. Some porins act as phage receptors and some are major antigens and exhibit pathogenic properties. Structurally, porins represent a new class of proteins, as they consist of a 16-stranded a-barrel. In contrast, the majority of membrane proteins is most likely a-helical. 2.1. Porin from Rhodobacter capsulatus Crystals of this porin have been first produced by Nestel et al. [41. They diffracted to about 3 A resolution. The structure of these crystals has been solved by multiple isomorphous replacement [5,6]. Since the crystallization failed for some period of time, the preparation procedure was revisited and modified. As a result, a new crystal form was found that diffracts to 1.8 A resolution and yields crystals routinely [7]. The major changes were the removal of SDS and EDTA from the preparation. Presumably, SDS had caused conformational heterogeneity and EDTA had removed the structurally important calcium ions [3]. After a peptide-based sequence analysis [81,the structure of this porin was solved at 1.8 A resolution by molecular replacement [3], yielding the coordinates of all 301 amino acid residues, 3 Ca2~ ions, 4 detergent molecules and 274 water molecules per subunit. See table 1 for abbreviations. The high resolution crystal form was obtained at 20°C,using the hanging drop method with 10 p.! drops containing 5 mg/ml protein in Tris at pH 7.2, 0.6% n-octyltetraoxyethylene, 0.3M LiC1 and 9% PEG-600. The reservoir solution was the
AK1, AK2, AK3 AKeco AKyst DTT Ap~A EDTA MES MOPS
Isoenzymes of mammalian adenylate kinases Adenylate kinase from E. co/i Adenylate kinase from yeast 1, P5-bis(adenosine-5’-)pentaphosphate PDithiothreitol Ethylenediaminetetraacetate Morpholinoethanesulfonate Morpholinopropanesulfonate
PEG SOS TPP Tris
Polyethyleneglycol Sodium dodecylsulfate Thiaminepyrophosphate Tris(hydroxymethyl)aminomethane
same but without porin and with 27% PEG-600. The space group is R3 (a b 92.3 A, c 146.2 A) with 1 subunit per asymmetric unit. As shown in fig. la, the crystal habit is rhombohedral with edge lengths of up to 600 p.m. =
=
=
2.2. Porin from Rhodopseudomonas blastica This porin is another member of the family of pore forming proteins in the outer membrane of Gram-negative bacteria. Like most other porins it forms trimers. Preliminary sequence data show only low homology (around 25% identical amino acids) with the porin from Rhodobacter capsulatus. For crystallization, we used the hanging drop method at 20°C. The drop contained 5 mg/ml protein, 20mM Tris at pH 6.8, 0.35M LiCl, 0.6% n-octyltetraoxyethylene and 17% PEG-600. The reservoir was at 35% PEG-600. Surprisingly, these conditions are almost the same as for the porin from Rh. capsulatus. The crystals grow routinely within about 2 weeks to a maximum size of 400 X 400 x 350 p.m3. The best crystals are nearly cubic in shape, as shown in fig. lb. The space group is R3 (a b 104.3 A, c 125.1 A) with 1 subunit per asymmetric unit. They diffract to at least 2.0 A. Native X-ray diffraction data to 2.3 A resolution have been collected on a Xentronics area detector installed on a Rigaku rotating anode tube. The structure analysis is in progress. =
=
=
388
G.E. Schulz et a!.
/ Highly ordered crystals
3. Crystallization of nucleoside-monophosphate kinases
of channel-forming membraneproteins
maximum size of 1000 X 800 X 600 p.m3. X-ray diffraction data were collected to 2.1 A resolution. In the case of double mutant Asp89 Val/ Arg165 —s Ile, the crystals grew within a few days to sizes around 500 x 300 X 100 p.m3. The most —*
These enzymes transfer a phosphoryl group onto a phosphoric acid forming an anhydride bond according to the reaction: Mg2~N 1TP + 2~+N N2MP N~DP+ Mg 2DP, where N1 and N2 are 2 nucleotide bases. Since this transfer is energetically much less favorable than the transfer to water forming orthophosphate, these kinases have to take great care to exclude water from their active center in order to avoid undesirable NTP hydrolysis, i.e. destruction of free energy. Given these conditions, the enzymes are remarkably small with Mr in the range 20,000 to 25,000. As a consequence, one has to expect large induced-fit movements during catalysis, which indeed have been observed [91. This obstructs soaking experiments with substrates as these are bound to break the crystals. Thus, all structural data on the catalytic mechanism have to be derived from cocrystais with ligands. We have analyzed a number of native and mutated enzymes of this group in free and in ligated forms. For each analysis, a new crystallization procedure had to be established. 3.1. Mutants of adenylate kinase from yeast ligated with substrates The structure of this enzyme has been solved some time ago [10]. Moreover, the AKyst gene has been cloned into E. coli to facilitate the preparation of large amounts of the protein and to carry out site-directed mutagenesis. A catalytic inactive double mutant (Asp89 —s Val, Arg165 —s lie) has been cocrystallized with ATP and AMP. Mutant 1le213 —s Phe has an exchange in the hydrophobic core decreasing its thermostability. This mutant has been crystallized with the twosubstrates-mimicking inhibitor Ap5A under essentially same,out, conditions the wildtype enzyme. It the turned however,as that this mutant
regularly shaped crystals are obtained by macroseeding and by the addition of KCI and f3-octylglucoside. Data were collected to 2.7 A resolution. Double mutant Asp89 2Val/Arg165 lIe 2~2 crystallized in space group P 1 1(a 48.2 A, b 73.4 A, c 119.8 A) with 2 molecules per asymmetric unit. The drop contained 50mM imidazole at pH 6.2, 21% PEG-3350, 2.5mM ATP and AMP, 10 mg/mI protein, 0.05% /3-octylglucoside and 75mM KC1. The reservoir was 50mM imidazole at pH 6.2 with 28% PEG-3350. In contrast, the wildtype enzyme and mutant 1le213 -s Phe crystallize in space group P1 (a 36.0 A, b 40.4 A, c 45.5 A, a 110.7°, /3 108.80, y 64.7°)with I molecule per asymmetric unit. In these cases the drop contained 60mM imidazole at pH 7.2, 26% PEG-8000, 2mM Ap5A, 12 mg/mi protein and (only for the mutant) 2mM /3-mercaptoethanol. The reservoir was 60mM imidazole at pH 7.2 with 29% PEG-8000. —,
—‘
=
=
=
=
=
=
=
=
=
3.2. Cocrystals of wildtype and mutant adenylate kinase from E. coli and inhibitor The structure of AKeco in complex with the two-substrate-mimicking inhibitor Ap5A has been solved [11]. The final R-factor is 19.6% at a resolution of 1.9 A. The structures of two mutants of AKeco were determined at 2.4 and 3.4 A resolution, respectively. For the crystallization of the wildtype enzyme, we used the hanging drop method at 20°C.The drop contained 22 mg protein/mI, 2mM Ap5A, 50mM MES at pH 6.7, 1.5% PEG-1500 and 1.5M ammonium sulfate. The reservoir had 50mM MES at pH 6.7 with 2.1M ammonium sulfate. Crystals grew within 3.4 days space to a maximum The group is size P2 of 1200 x 500 x 150 p.m 1221 (a 73.2 A, b 79.8 A, c 84.8 A) with 2 molecules per asymmetric unit. A substantial improvement with respect to size and reproducibility of the crystals was achieved by addition of PEG-1500, which re=
required /3-mercaptoethanoi for crystal growth. Crystallization was carried out at 20°C using the hanging drop method. With mutant 1le213 —s Phe, the crystals grew within a few days to a
=
=
G.E. Schulz eta!.
/ Highly ordered crystals of channel-forming membrane proteins
389
duced the tendency of the crystals to grow attached to each other. Mutant GlylO —s Va! crystallizes under wild-
unit. The crystals diffract to at least 1.9 A resolution. The structure has been solved and is currently being refined. The minor component AK22
type conditions in the same space group, but with a c-axis prolonged by 1.5%. The mutant crystals tend to grow bigger in size than the wildtype crystals. Mutant Pro9 —s Leu yielded only thin crystal plates under wildtype crystallization conditions. By addition of 2% ethanol, we could obtain a new crystal form that diffracts reasonably well. The space group is P21 (a = 60.3 A, b 77.8 A, c 57.7 A, /3 = 94.3°)with 2 molecules per asymmetric unit. Wildtype AKeco and mutant Pro9 Leu were also crystallized under microgravity conditions. Both proteins were sent into space using an unmanned Chinese re-entry system and were incubated there for 8 days (COSIMA-I). This experiment yielded crystals of the wildtype and the mutant enzyme. Size and quality of these crystals, however, did not reach the quality of the crystals grown in the home laboratory [1].
crystallized under similar conditions. The obtamed crystals, however, are not yet suitable for X-ray analysis.
=
=
—,
3.3. Mitochondrial adenylate kinase In mammalian cells there are three adenylate kinase isozymes: AK1 in the cytosol, AK3 in the mitochondrial matrix and AK2 in the mitochondna! intermembrane the structures of AK1 [12] and AK3space. [131 While have been solved at high resolution, AK2 has resisted crystallization for many years. Recently, we discovered that the enzyme preparation consists of 2 variants, AK2 1 and AK22, that differ in 3 amino acids from each other. After separating these species on large scale by chromatofocussing, we succeeded to grow crystals from both of them using the hanging drop method at 20°C.After macroseeding, the major component AK21 grew within 5 days 3. to A maximum crystal is sizes around 1500 500 drop x 180contained p.m depicted in fig. ic. XThe 7 mg/ml enzyme, 20mM MOPS at pH 7.1, 5mM /3mercaptoethanol, 5mM MgSO 4 and 9% PEG3350. The reservoir was 20% PEG-3350, 5mM f3-mercaptoethanol in 20mM MOPS at pH 7.1. The space group is P2~2~21 (a = 44.3 A, b = 50.1 A, c = 122.3 A) with 1 molecule per asymmetric
3.4. Guanylate kinase ligated with GMP Guanylate kinase resembles the adenylate kinases with respect to size and reaction mechanism. The enzyme from yeast has been crystallized in complex with its substrate GMP and the structure has been determined at 2.0 A resolution [14]. Two crystal forms were obtained. Crystal form I belongs to space group P432~2(a b 50.8 A, c 155.3 A) with 1 molecule per asymmetric unit. Crystal form II has space group C2221 (a = 71.2 A, b = 72.3 A,. c = 152.7 A) with 2 molecules per asymmetric unit. Both crystals forms grow at the same conditions and can be interconverted by temperature changes: form I predominates at 4°Cwhereas form I and form II occur equally distributed at 25°C, even in one drop. The interconversion corresponds to small changes in the molecular packing. Using the hanging drop method, crystals grew within about 3 days 3,upastoshown a maximum sizeThe of in fig. ld. 1200 X 600 X 400 p.m crystal size increased dramatically when PEG1000 was added until phase separation occurred. The drop contained 7 mg/ml enzyme, l.OM am=
=
=
monium sulfate, 1mM GMP, 1% PEG-bOO, 40mM potassium phosphate at pH 5.5. The reservoir was 2.OM ammonium sulfate, 40mM potassium phosphate at pH 5.5. During the structure analysis all crystal handling had to be done in the mother liquor, since no enzyme-free stabilizing solution could be found. 3.5. Uridylate kinase Uridylate kinase from yeast is homologous (40% sequence identity) to AK1. It was crystallized using the hanging drop method with PEG as precipitant. The addition of up to 0.025% /3-octylglucoside prevented the formation of microcrystalline material and increased the crystal size
390
G.E. Schulz et a!.
/ Highly ordered crystals of channel-forming membrane proteins
up to a maximum size of 700 X 300 x 300 p.m3. The space group is P6 1522 (a = b 64.2 A, c = 185.0 A) with one molecule per asymmetric unit. The crystals diffract to at least 2.1 A resolution. The structure analysis is under way. =
4. Crystallization of flavo-enzymes In addition to human glutathione reductase [15] which has now become a model enzyme for the increasing family of FAD-containing oxidoreductases, several other flavo-enzymes are now investigated in our laboratory. We are particularly interested in the reaction mechanisms and evolutionary relationships of these enzymes. Recently a scientifically and technically important mutant of glutathione reductase that changed the cofactor specificity from NADP to NAD [16] attracted widespread attention. We are currently analyzing this change in order to explain it structurally. 4.1. Wildtype and mutant glutathione reductase from E. coli The enzyme reduces glutathione at the expense of NADPH. It consists of 2 identical subunits with Mr = 48,700 each. Its structure is known at 3 A resolution [17]. Using the hanging drop method, the enzyme was crystallized in 3 different forms, 2 of which were suitable for X-ray analysis.crystals grow in drops containing Form-PlO 5% PEG-10000 (Fluka), 20mM potassium phosphate at pH 5.4 and 15 mg/rn! protein. The reservoir had 20% PEG-10000, 20mM potassium phosphate at pH 5.4. The maximum crystal size was 2500 x 800 x 800 p.m3. The space group is P4 3212 (a = b = 62.0 A, c = 336.5 A) with 1 subunit per asymmetric unit. The crystals diffract anisotropicall~’to 3.0 A resolution along the c-axis and to 3.4 A resolution along a and b. The anisotropic diffraction of form-P 10 seems to be explained by the few observed contacts in the a—b plane. Therefore, we attempted to improve the packing order by site-directed mutagenesis
inserting a new contact in this plane. The crystallization of this mutant enzyme is in progress. Form-P8 crystals grow in drops of 7% PEG8000 (Sigma), 100mM potassium phosphate at pH 5.5, 20 mg/mI protein using a reservoir of 20% PEG-800 and 100mM potassium phosphate at pH 5.5. After macnoseeding with 14% crystals size PEG50 X 3 (washed with andof7% 8000 solutions), we obtained crystals with a maxi50 X 50 p.m mum size of 800 X 400 X 300 p.m3. The space group is P2
1 (a = 120.5 A, b = 73.5 A, c = 60.5 A, yThe = 82.7°)with 2 subunits per asymmetric unit. crystals diffract to 2.0 A resolution. Form-P8 is very difficult to reproduce without seed crystals. Only about 1% of the crystallization attempts were successful. 4.2. NADH peroxidase NADH peroxidase is a tetrameric flavo-enzyme (symmetry D2) with an Mr of 50,300 per subunit. The enzyme catalyzes the degradation of H202 at the expense of NADH, FAD and an unusual stabilized cysteine-sulfenic acid participate in catalysis. Normally, H2O2 reduction is catalyzed by heme- or selenium-containing proteins. NADH peroxidase crystallizes in the space group 1222 (a = 77.2 A, b = 134.5 A, c = 145.9 A). The crystals diffract to 2.1 A resolution and contain 1 subunit per asymmetric unit. The structure has been determined [18]. Using the hanging drop method, crystals grew within 3 days up toofa 3. Most maximum size of 1000 x 500 X 300 p.m them were of flowerlike shape. The drop contamed 5 mg/rn! enzyme, 1.05M ammonium sulfate, 2mM DTT, 2.5 p.M FAD, and 50mM potassiurn phosphate at pH 7.0. The reservoir was 2.1M ammonium sulfate, 2mM DTT, 5p.M FAD and 50mM potassium phosphate at pH 7.0. 4.3. Pyruvate oxidase The enzyme from Lactobacillus plantarum is a homotetramer with an Mr of 263,000. It contains the coenzymes FAD and TPP. For crystallization we applied the hanging drop method. The enzyme crystallizes readily in form A using 20mM
G.E. Schulz et al.
/ Highly ordered crystals of channel-forming membrane proteins
phosphate at pH 5.7—6.5, 20 mg/ml protein, O.5M ammonium sulfate in the drop, and the same buffer with 1M ammonium sulfate in the reservoir. The average crystal size was 700 x 700 x 300 p.m3. Because of its extremely high mosaicity, crystal form A is not suitable for X-ray analysis. After a tedious search, we could eventually produce crystals of form B using drops with 20mM phosphate at pH 5.2, 1mM TPP, 20 mg/ml protein, O.5M ammonium sulfate and 5% glycerol. The reservoir contained the same buffer and 1.OM ammonium sulfate with 10% glycerol. Crystals grow within 6 months to a maximum size of 1000 x 90 x 90 p.m3 in a small percentage of the drops. The crystals are well ordered and diffract to beyond 2.4 A resolution. The space group is C222 1 (a = 121.3 A, b = 154.7 A, c = 166.7 A) with analysis 2 subunits asymmetric unit. The structure is inper progress.
5. Crystallization of sugar-processing enzymes This group of enzymes is most important for the food and drug industry. One may expect future applications in other parts of the chemical industry because saccharides are among the cheapest organic raw materials. We are analyzing cyclodextrin glycosyltransferase, which catalyzes the conversion of starch to cyclodextrins, widely used as micro-encapsulators. Furthermore, we work on an aldolase which may become important for the production of defined chiral cornpounds as building blocks for organic synthesis [19]. Here, we intend to change substrate specificities by site-directed mutations. 5.1. Cyclodextrin glycosyltransferase The enzyme from Bacillus circulans is monomeric and consists of 684 amino acid residues. The structure of the wildtype enzyme was refined at 2.0 A resolution with an R-factor of 17.6% [20]. Nine mutants (Pro138 —s Ala, Tyrl9l —s Phe, Tyr195 Phe, Asp229 —s Ala, Phe259 —s Leu, Ser276 —s Cys, Asp328 —s Ala, Met329 —s Asn and Ser428 —s Cys) were crystallized isomorphously with the wildtype enzyme. ~
—
391
The Cys-mutants yielded useful mercury derivatives that improved the multiple isomorphous replacement electron density map. The structures of mutants Pro138 —s Ala, Asp229 Ala, Phe259 Leu, Asp328 Ala and Met329 —s Asn were refined at 2.5 and 2.7 A resolution. The wildtype enzyme crystallizes in batches over time spans of 3 to 12 months. For the mutant enzymes we used hanging drops at similar conditions as with the wildtype, but macroseeding was done with wildtype crystals. The drops contamed 0.6M ammonium sulfate, 50mM tnethanolamine, 50mM citric acid at pH 6.7, 0.5% PEG-1500 and 11 mg/mI protein. The reservoir was 0.9M ammonium sulfate, 50mM tnethanolamine, 50mM citric acid at pH 6.7 and —
—‘
—
0.5% PEG-l500. The obtained 3maximum crystal for the mutants, sizes 3000 werex1500 100 for p.mthe wildtype. One and 300 xx 150 300 xp.m3 example is shown in fig. le. The crystal space group is P2 12121 (a =94.8 A, b=104.7 A, c= 114.0 A) with 1 molecule per asymmetric unit. The diffraction pattern extends to 1.9 A resolution. 5.2. Fuculose-1-phosphate aldolase enzyme from E. coli and belongs to Zn the2~for class II The (metal binding) aldolases needs catalytic activity. The native enzyme is a homotetramer with an Mr of 24,000 per subunit. It is prepared from an overexpressing strain by a three-step purification consisting of ion exchange, hydrophobic interaction and gelpermeation chromatography. For crystallization we used the hanging drop method at 20°Cand ammonium sulfate as a precipitant. Within about 10 days crystals with a maximum size of 500 x 500 x 110 p.m3 were obtained. They belong to space group P42 12 and diffract to a resolution of 2.2 A. They contain one subunit per asymmetric unit. The structure analy. sis is in progress. 6. Conclusions Today the crystallization problem is obviously different from a decade ago, the most drastic
392
G.E. Schulz el a!.
/ Highly ordered crystals of channel-forming membrane proteins
change being the availability of large amounts of recombinant proteins. Having enough material, a large number of crystallization parameters can be screened conveniently. But even more important is the dramatic shortening of the isolation procedures for recombinant proteins. While purification factors of the order of a million are required to isolate a protein from natural living material, this factor is reduced to the order of a hundred in reasonably well-expressed recombinant proteins. Cutting the isolation procedure short means reducing the strain on the protein, which leads to conformationally more homogeneous proteins that crystallize more readily. We also find that conformational homogeneity can often be enhanced by cocrystallization with protein ligands. Taken together, one may expect that in the future crystallization will become less and less of an obstructing road block in structure analyses.
References [I] V.A. Erdmann, C. Lippmann, C. Betzel, Z. Dauter, K. Wilson, R. Hilgenfeld, J. Hoven, A. Liesum, W. Saenger. A. Miiller-Fahrnow, W. Hinrichs, M. DOvel, G.E. Schulz, C.W. MOller, HG. Wittmann, A. Yonath, G. Weber, K. Stegen and A. Plaas-Link, FEBS Letters 259 (1989) 194. [2] R.M. Garavito and J. Rosenbusch, J. Cell Biol. 86 (1980) 327.
[3] M.S. Weiss, A. Kreusch, E. Schiltz, U. Nestel, W. Welte, J. Weckesser and G.E. Schulz, FEBS Letters 280 (1991) 379. ~ U. Nestel, T. Wacker, D. Woitzik, J. Weckesser, W. Kreutz and W. Welte, FEBS Letters 242 (1989) 405. [5] MS. Weiss, U. Nestel, T. Wacker, J. Weckesser, W. Kreutz and G.E. Schulz, FEBS Letters 256 (1989) 143. [6] M.S. Weiss, T. Wacker, J. Weckesser, W. Welte and G.E. Schulz, FEBS Letters 267 (1990) 268. [7] A. Kreusch, MS. Weiss, W. Welte, J. Weckesser and G.E. Schulz, J. Mol. Biol. 217 (1991) 9. [8] E. Schiltz, A. Kreusch, U. Nestel and G.E. Schulz, European J. Biochem. 199 (1991) 587. [9] G.E. Schulz, C.W. Muller and K. Diederichs, J. Mol. Biol. 213 (1990) 627. [10] U. Egner, AG. Tomasselli and G.E. Schulz, J. Mol. Biol. 195 (1987) 649. [11] C.W. Muller and G.E. Schulz, J. Mol. Biol. 202 (1988) 909. [12] D. Biol.Dreusicke, 199 (1988) PA. 359. Karplus and G.E. Schulz, J. Mol. [13] K. Diederichs and G.E. Schulz, J. Mol. Biol. 217 (1991) 541. [14] T. Stehle and G.E. Schulz, J. Mol. Biol. 211 (1990) 249. [15] PA. Karplus and G.E. Schulz, J. Mol. Biol. 210 (1989) 163. [16] N.S. Scrutton, A. Berry and R.N. Perham, Nature (London) 343 (1990) 38. [17] U. Ermler and G.E. Schulz, Proteins: Struct. Funct. Genet. 9 (1991) 174. [181 T. Stehle, S.A. Ahmed, A. Claiborne and G.E. Schulz FEBS Letters 267 (1990) 186. [19] W.-D. F~ssner,G. Sinerius, A. Schneider, M. Dreyer, G.E. Schulz, J. Badia and J. Aguilar, Angew. Chem. Intern. Ed. EngI. 30 (1991) 555. [20] C. Klein and G.E. Schulz, J. Mol. Biol. 217 (1991) 737.