Progress in the Development of an Alternative Approach to

CRYSTAL GROWTH & DESIGN

Progress in the Development of an Alternative Approach to Macromolecular Crystallization

2008 VOL. 8, NO. 8 3038–3052

S. B. Larson,† J. S. Day,† C. Nguyen,‡ R. Cudney,‡ and A. McPherson*,† Department of Molecular Biology & Biochemistry, Steinhaus Hall, Room 560, UniVersity of California, IrVine, California 92697-3900, and Hampton Research, 34 Journey, Aliso Viejo, California 92656-3317 ReceiVed February 15, 2008; ReVised Manuscript ReceiVed March 29, 2008

ABSTRACT: We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, noncovalent cross-links in crystals, and thereby promote lattice formation. Earlier experiments provided encouraging results that suggested further research was warranted (Larson, S. B.; Day, J. S.; Cudney, R.; McPherson, A. A novel strategy for the crystallization of proteins: X-ray diffraction validation. Acta Crystallogr., D: Biol. Crystallogr. 2007, 63, 310-318. McPherson, A.; Cudney, B. Searching for silver bullets: an alternative strategy for crystallizing macromolecules. J. Struct. Biol. 2006, 156, 387-406). Here we report additional, large-scale crystallization screening experiments that lend further support, though they suggest that additional mechanisms may play a positive role as well. As before, we accompanied the crystallization experiments with X-ray diffraction analyses of some of the crystals grown. A number of these showed incorporation of conventional molecules into protein crystal lattices, and further validated the underlying hypothesis. The strategy we are pursuing is essentially orthogonal to current approaches and has an objective of doubling the success rate of today. Introduction In earlier papers1–3 we advanced the idea of an alternative approach to the crystallization of macromolecules based on the inclusion in mother liquors of conventional small molecules, formulated as cocktails, that would promote lattice formation. They would do so by (a) stabilizing, or altering the conformation of the protein, (b) perturbing the interaction of the protein with the solvent, or most importantly, (c) by participating in building the lattice by forming reversible cross-links between macromolecules in the crystal. In those papers we described three separate experiments where we attempted to crystallize 81 different proteins and viruses using mixtures of about 200 different chemical compounds. Only two fundamental crystallization conditions were otherwise used. These were 30% PEG 3350 at pH 7, and 50% Tacsimate at pH 7. The results of the experiments were encouraging. More than twice as many proteins were crystallized overall as were crystallized from controls free of any small molecules. In addition, there were frequent occasions where some exceptional result was obtained for specific proteins, and numerous examples where new or unusual crystal forms were obtained. Some of the crystals grown in the experiments were subsequently analyzed by X-ray diffraction to determine if small molecules could be visualized in the crystal lattice. Nine examples were presented where this was indeed the case, where small molecules were at the centers of hydrogen bonding networks linking two or more protein molecules in the lattice.1 An independent analysis was also performed on one of the crystals from the experiments, and similar results were reported.4 The X-ray diffraction results essentially validated the underlying hypothesis. From the earlier work some chemical compounds and groups of compounds emerged as attractive possibilities for inclusion * Corresponding author. Tel: (949) 824-1931. E-mail: [email protected]. † University of California. ‡ Hampton Research.

in cocktails. For example, both alkyl chains and aromatic moieties carrying two or more carboxyl groups often promoted crystal growth, as did those compounds bearing multiple amino groups, or combinations of carboxyl and amino groups. The principal requirement seemed to be that the compound had the ability to engage in multiple, strong hydrogen bonds with the protein. In some cases, hydrophobic interactions between small molecules and proteins were also seen to be important. As we pointed out in the earlier papers, however, it remains unclear exactly which small molecules provide the greatest chance of success in general, and it is certainly unknown which might best serve a specific protein. The best that we can hope is that we can ultimately compile a library of potentially useful conventional molecules that can in turn be formulated into effective crystallization promoting cocktails. The entries in that library will have to be, we believe, laboriously sought by carrying out experiments like those we described previously2,3 and those described here. The experiments that we present in this paper are similar to those reported earlier, using many of the same macromolecules in an augmented test set, but employing a different array of conventional small molecules. From these experiments we can enlarge the library of helpful compounds, and obtain additional clues as to which classes of small molecules deserve further attention. In addition, we present selected X-ray diffraction studies of some of the crystalline proteins that further demonstrate the validity of the idea, and shed additional light on the essential properties of the most useful small molecules. Experimental Section Crystallization Experiments. The crystallization screening methods were essentially the same as those described previously,2 with the following changes. In the former experiments, two fundamental crystallization conditions were used, one based on PEG 3350, and the other on 50% Tacsimate. In the current experiments, only the PEG condition was used. The initial starting concentration of PEG in the crystallization drop was here 5%. In these experiments, all drops were

10.1021/cg800174n CCC: $40.75  2008 American Chemical Society Published on Web 07/03/2008

Approach to Macromolecular Crystallization 2 µL volume and were dispensed by a Phoenix crystallization robot (A. J. Robbins, Sunnyvale, CA) into 96 well Intelliplates (Hampton Research, Aliso Viejo, CA). The reservoirs were 90 µL of 25% PEG 3350. Samples were examined at three-week intervals by the investigators using an Olympus SZX12 microscope with polarized light. The samples were tracked for 5 months. The proteins used in the experiments, shown in Table 1, are for the most part those used in the earlier experiments,2 with some substitutions and additions. When not in use, they were maintained at -80 °C over the course of the investigation. Included in the experiments were 7 tRNAs, and the genomic RNA from satellite tobacco mosaic virus (STMV). The purpose of their inclusion was to determine whether the approach we propose is applicable to nucleic acid molecules as well as to proteins and viruses. The plates containing the protein and virus crystallization trials were stored at 22 °C after dispensing of the drops and sealing with clear plastic tape. The plates containing the RNA molecules were stored at 4 °C. We will refer to the two experiments presented here as Experiment IV and Experiment V, since they are extensions of the earlier experiments.2 In Experiment IV, 96 mixtures of small molecules, or cocktails, were investigated for their propensity to promote crystallization of the proteins shown in Table 1. All Experiment IV cocktails were buffered with 0.1 M HEPES and adjusted, before use, to pH 7 as necessary. The cocktails for Experiment IV are shown in Table 2. An inspection of Table 2 shows that there are five principal categories of compounds that were investigated, and these were (1) histological dyes and stains known to bind to biological macromolecules, (2) antibiotics and drug molecules known to specifically interact with enzymes, (3) peptides, oligosaccharides, and oligonucleotides, both synthetic and produced from natural polymers digested with hydrolytic enzymes, (4) various nucleotides, and (5) a rather arbitrary collection of small organic molecules, most based on an aromatic moiety. Experiment V was more ambitious, and required two 96 well plates per protein, or 192 total samples. In this experiment, 62 different small molecule cocktails were used, and these are also shown in Table 2. The cocktails, in this case, however, were made in triplicate, with one at pH 5.8, the second at pH 7.0, and the third at pH 8.2. Thus every cocktail in experiment V was investigated at three different pHs. The cocktails were buffered with 0.1 M MES, HEPES, and Tris-HCl respectively. The reservoirs were similarly buffered according to the sample with which they equilibrated. In terms of robotic operations, in Experiment IV, there were two components to a sample drop, the protein and the cocktail containing PEG 3350. In Experiment V there were three components to each drop, the protein, the cocktail, and the buffered PEG 3350 reservoir. Again, the crystallization trials were scored manually by the investigators as above. In these experiments, we took a broader view of scoring the results, feeling that the earlier approach of simply scoring samples as having produced crystals or not were too rigorous. That kind of analysis ignored the many other kinds of useful observations that emerged from the crystallization trials. Evaluating the results of large arrays of crystallization trials is perhaps the most important part of an investigation. Again, it is important to emphasize that over time, as experience with a large set of test proteins accumulates, certain macromolecules tend to stand out as meaningful indicators of positive or negative effects. Horse hemoglobin and beef catalase, for example, are not very helpful because they crystallize so readily. Yeast hexokinase, rabbit aldolase, and rabbit serum albumin are useful, because, in general, they do not readily crystallize. It seems evident, then, that greater weight should be given to successes with the latter proteins than to the former. Although we have now carried out a reasonable number of experiments with our macromolecule test set, we are still not to the point where we are ready to assign a quantitative, statistical weight to each protein. Thus, even in these experiments our judgment of what constitutes an exceptional event remains only semiquantitative, with, admittedly, a significant subjective component. Our current ideas on what is meaningful and what is not in evaluating the results of a crystallization experiment, like those described here, and what we take as strong and weak indicators have been discussed elsewhere.3 X-ray Diffraction Analyses. X-ray diffraction analyses of crystals grown in Experiments IV and V were carried out using the same methods as those described for the earlier experiments.1 The data were collected on crystals from these experiments, however, from flash frozen crystals rather than at room temperature as was previously the case. When necessary, a solution consisting of 20% PEG 3350 and 30%

Crystal Growth & Design, Vol. 8, No. 8, 2008 3039 Table 1. Experiments IV and V symbol

protein

ALD APHS APS ATRF BA BAMY BC BJ-KWR BJ-MLE BLCM BLCT BLIP BMV BT BYAD CK CLIPs CNV ConA ConB CT CTSY CYTC D FE GI GPDH GRM HEX HG HH HMCY HP HS HuH I IDEC151 INV LDH LFN LtRNA LYS MAB231 MLC MYG NL OVL PA PAP PEP PHS PK PMV PRX PT RA RB RNN RSA SBTI SJL SOD SPMV STMV THM TRX TYMV UBQ XYL yptRNA RLC β-LG

aldolase, rabbit muscle prostatic acid phosphate, bovine alkaline phosphatase, bacterial apotransferrin, bovine milk alpha amylase, bacterial beta amylase, sweet potato catalase, bovine Bence Jones Protein-K.W.R., human Bence Jones Protein-M.L.E., human beta-lactamase, bacterial catalase, bacterial lipase, bacterial brome mosaic virus protein trypsin-benzamidine, bovine alcohol dehydrogenase, yeast creatine kinase, rabbit muscle lipase, Candida canavalin, Jack bean concanavalin A, Jack bean concanavalin B, Jack bean alpha chymotrypsin, bovine citrate synthase cytochrome C, horse heart deoxyribonuclease, bovine ferritin glucose isomerase, bacterial glyceraldehyde phosphate dehydrogenase gramacidin, synthetic hexokinase, yeast hemoglobin, goat hemoglobin, horse hemocyanin, keyhole limpet hemoglobin, pig hemogobin, ovine hemoglobin, human insulin, bovine monoclonal Ab IDEC151 invertase, yeast lacate dehydrogenase, rabbit muscle lactoferrin, bovine milk leucine tRNA lysozyme, hen egg monoclonal Ab MAB231 catalase, Micrococcus lutens myoglobin, horse lipase, Thermomyes lanuginosa ovalbumin alpha-amylase, pig papain, papaya pepsin, bovine phaseolin, kidney bean proteinase K, fungal Panicum Mosaic Virus, millet peroxidase, horseradish trypsin-benzamidine, porcine ribonuclease A, bovine ribonuclease B, bovine rennin, cow serum albumin, rabbit trypsin inhibitor, soy bean lectin, Saphora japonica superoxide dismutase satellite Panicum mosaic virus, millet satellite tobacco mosaic virus thaumatin, serendipity berry xylanase, fungal turnip yellow mosaic virus ubiquitin, bovine erythrocytes xylanase, bacterial Phe tRNA, yeast alpha lactalbumin, bovine milk beta lactoglobulin, bovine milk

MPD in water was generally adequate for cryoprotection. Use of cryotechniques allowed us to investigate crystals of smaller size. The

3040 Crystal Growth & Design, Vol. 8, No. 8, 2008

Larson et al. Table 2 Experiment Va

Experiment IV 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Methyl orange, quinine, fructose 1,6 diphosphate Fusidic acid, mixture of multi phosphates Crystal violet, gibrillic acid, uridine-2′,3′-cyclic monophosphate Ellipticine, acriflaven, ethidium bromide, 9-bromoanthracene Coomassie brilliant blue, digitonin, TPCK Br-, I-, BO33-, PO43-, NO3-, ReO4Basic fuchsin, streptomycin, quinine Cystine, β-mercaptoethanol, dithio erythritol, dithiothreitol, glutathione Safranin orange, cordycepin, chlorpromazine Daunomycin, ADP, pentaglycine Chlorophenol red, novobiocin, β-glycerophosphate GTP, UTP, CTP, ATP Cresol purple, folinic acid, oubain Congo red, D-mannoheptulose, phenylglyoxal Hemin, puromycin aminonucleoside, mixture of polyphosphates Gentamycin, malachite green, fructose 1,6 diphosphate Toluidine blue, trimethoprim, β-glycerophosphate Dehydro isoandrosterone, UTP, CMP Colloidal silver, PEG-succinate, tetracycline Phosphorous acid triisodecyl ester, PEG-monooleyl ether, dactinomycin Casein/subtilisin digest, DNA/DNase digest, eosin blue Starch/amylase digest, casein/trypsin digest, pyronin Y Methylene blue, phenobarbital, ampicillin Tannic acid, mycophenolic acid, 3,5-dinitrosalicylic acid

1 4

25 26 27 28 29 30 31 32 33

Xylene cyanol, chlorpromazine, mycophenolic acid Erythromycin, trigalacturonic acid, d(pA)4 Thioflavin T, neomycin, pyridine-2-aldoximine Oubain, TPCK, d(pA)4 Azure bromide, distamycin, phosphatidyl choline 5,5-Diphenylhydantoin, digalacturonic acid, IMP Phenolphthalein, lincomycin, 5,5-diphenylhydantoin Theophylline, ruthenium red, n-acetyl-R-galactosamine Methyl green, methyl prednisolone succinate, tilorone

52 55 58 61 64 67 70

7 10 13 16 19 22 25 28 31 34 37 40 43 46 49

73

34 Erythromycin. Ampicillin, streptomycin, tetracycline 35 Remazol blue R, actinomycin D, phenobarbital 36 ONPG, AMP, quinaldine red

76 79 82

37 Bromphenol blue, tobramycin, 5,5-diphenylhydantoin

85

38 39 40 41 42 43 44 45 46 47

88

48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

IPTG, GMP, pyronin Y Colloidal gold, bactotryptone PEG-sterylamine, acarbose, rifampicin RNA/RNase A digest, casein/pepsin digest, glycogen/amylase digest Casein/proteinase K digest, cefotaxime, ATP RNA/RNase A digest, glycogen/amylase digest, pepstatin Casein/trypsin digest, casein/proteinase K digest, phosphatidyl choline DNA/DNase digest, starch/amylase digest, malachite green Bactotryptone, chloramphenicol d(pA)4, mixture of polyphosphates, methylene diphosphonic acid, fructose 1,6 diphosphate Control Quinaldine red, kanamycin, trimethoprim IPTG, GDP, mycophenolic acid Safranin orange, gibrillic acid, benzidine β-Octyl glucoside, heptanetriol, phosphatidyl choline, palmitic acid, batyl alcohol Methyl orange, novobiocin, IPTG D-Mannoheptulose, rifampicin, colloidal silver Toluidine blue, 5,5-diphenylhydantoin, hippuric acid Trigalacturonic acid, acarbose, tannin Coomassie brilliant blue, ampicillin, phenobarbital Trigalacturonic acid, TPCK, IMP p-Nitrophenylphosphate, p-benzoquinone, 3,3-diaminobenzidine CMP, AMP, GMP, UMP, IMP Remazol blue, cefotaxime, penicillin G Ethidium bromide, pentaglycine, methylene diphosphonic acid 9-Bromophenanthracene, chloramphenicol, GMP Dehydro isoandrosterone, U-2′,3′-cyclic phosphate, 9-bromophenanthracene 3,3′-Diaminobenzidine, acarbose, hippuric acid ONPG, digalacturonic acid, eosin blue Thioflavin T, gentamycin, p-nitrophenylphosphate Pyridine-2-aldoximine, GDP, Congo red

91 94 97 100 103 106 109 112 115 118

Anthrone, phenylglyoxal, cystamine dichloride 3,5-Dinitrosalicylic acid, 2,5-pyridinedicarboxylic acid, 1,5 naphthalenedisulfonic acid Congo red, sulfanilamide, salicin Sulfaguanidine, benzidine, nicotinamide Asp-leu, asp-asp-asp-asp Dithioerythritol, (()-carnitine hydrochloride, N,N,N′,N′,N′′-pentakis(2-hydroxypropyl)diethylenetriamine 1,3,5-Pentanetricarboxylic acid, benzoic acid, N,N,N′,N′,N′′-pentakis(2-hydroxypropyl)diethylenetriamine 3,5-Dinitrosalicylic acid, aspartame, 4-aminobenzoic acid Sulfosalicylic acid, 4-nitrobenzoic acid, naphthalene-1,3,6-trisulfonic acid trisodium salt Hippuric acid ammonium salt, sulfanilic acid, azelaic acid N-(2-Acetamido)-2-aminoethanesulfonic acid, poly(3-hydroxybutyric acid), benzidine Phenol, caffeine, nicotinamide 2,7-Naphthalenedisulfonic acid disodium salt, 2,6-Naphthalenedisulfonic acid disodium salt 1,5-Naphthalenedisulfonic acid disodium salt, naphthalene-1,3,6-trisulfonic acid trisodium salt 3,5-Dinitrosalicylic acid, sodium 4-aminosalicylate, salicylamide Salicin, caffeine, 2′-deoxyguanosine-5′-monophosphate L-Tyrosine, L-phenylalanine, L-tryptophan, L-histidine, L-isoleucine, L-leucine Urea, caffeine, guanidine hydrochloride Phenol, L-glutamic acid, trimethylamine N-oxide 4-Nitrobenzoic acid, 2,5-pyridinedicarboxylic acid, mellitic acid Sulfaguanidine, phenylglyoxal, sulfanilamide Benzidine, hippuric acid ammonium salt, salicylic acid Anthrone, N-(2-acetamido)-2-aminoethanesulfonic acid, Congo red N,N,N′,N′,N′′-Pentakis(2-hydroxypropyl)diethylenetriamine, dithioerythritol, 2′-deoxycytidine-5′-monophosphate Poly(3-hydroxybutyric acid), 1,3,5-pentanetricarboxylic acid, trimesic acid Cystathionine, L-citrulline, cystamine dihydrochloride Azelaic acid, vanillic acid, trans-cinnamic acid 5-Sulfosalicylic acid, 3-aminobenzenesulfonic acid, 3,5-dinitrosalicylic acid trans-Cinnamic acid, 2,7-naphthalenedisulfonic acid disodium salt, azelaic acid 2-Aminobenzenesulfonic acid, 1,3-benzenedisulfonic acid disodium salt, 2,6-naphthalenedisulfonic acid disodium salt 5-Sulfoisophthalic acid monosodium salt, trans-cinnamic acid, 1,4-cyclohexanedicarboxylic acid 3-Aminobenzoic acid, sodium 4-aminosalicylate, salicylic acid Salicylamide, vanillic acid, sulfanilamide Phenylurea, p-coumaric acid, sulfaguanidine 1,2-Diaminocyclohexane sulfate, 1,4-cyclohexanedicarboxylic acid, methylenediphosphonic acid L-O-Phosphoserine, O-phospho-L-tyrosine, 6-phosphogluconic acid trisodium salt Methylenediphosphonic acid, sodium triphosphate pentabasic, 2′-deoxyadenosine-5′-monophosphate Benzamidine hydrochloride, L-ornithine, (()-carnitine hydrochloride L-(+)-Canavanine sulfate monohydrate, cystathionine, DL-cystine Ala-gly, ala-his, gly-leu

121 124 127 130 133 136 139 142 145 148

Gly-gly, lys-lys-lys-lys, asp-leu Leu-gly-gly, gly-gly-gly, leu-gly Gly-phe, asp-asp-asp-asp, L-(+)-canavanine sulfate monohydrate Ala-ala, ala-his, gly-asp Leu-gly, gly-phe, lys-lys-lys-lys-lys Ala-gly, leu-gly, benzidine Gly-phe, DL-cystine, ala-his Gly-asp, gly-gly-gly-gly Leu-gly-gly, ala-ala, gly-gly 2-Sulfobenzoic acid hydrate, 5-sulfosalicylic acid, 2′-deoxyuridine-5′-monophosphate disodium salt 151 Cystamine dihydrochloride, aspartame, benzamidine hydrochloride 154 Tris (hydroxymethyl)aminomethane, HEPES, CHAPS 157 MES monohydrate, PIPES, BICINE 160 Gadolinium(III) chloride hexahydrate, benzamidine hydrochloride, 2′-deoxycytidine-5′-monophosphate, guanidine hydrochloride,

Approach to Macromolecular Crystallization

Crystal Growth & Design, Vol. 8, No. 8, 2008 3041 Table 2. Continued

Experiment IV 69 70 71 72 73 74 75 76 77 78 79

Chlorophenol red, phenobarbital, salacin Neomycin, ADP, Congo red Crystal violet, cordycepin, penicillin G Sorbitol, glycerol, MPD, hexanediol, ethylene glycol, propylene glycol Phenolphthalein, 2,4-dinitrosalicylic acid, phenylglyoxal Methyl prednisolone succinate, UTP, dactinomycin Distamycin, d(pA)4, oubain β-Octyl glucoside, 1,2,3, heptanetriol, tetracycline, phenyl boronic acid Methyl green, quinine, GDP Chlorpromazine, ruthenium red, methylene diphosphonic acid Azure bromide B, erythromycin, 3,3′ diamino benzidine

80 Tobramycin, UMP, basic fuchsin 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Cresol purple, n-acetyl-R-galactosamine, kanamycin Tobramycin, digitonin, p-benzoquinone RNA/RNase A digest, bactotryptone (2×) Fusidic acid, 1,2,3-heptanetriol, pepstatin Casein/subtilisin digest, starch/amylase digest, salacin Casein/proteinase K digest, daunamycin, pentaglycine All of the digests, actinomycin D Bactotryptone, 3,5-dinitrosalicylic acid, puromycin nucleotide pnpa-gln Colloidal silver, bactotryptone, colloidal gold PEG-stearylamine, ellipticine, phosphatidyl choline Phosphorous acid triisodecyl ester, PEG-monooleyl ether, colloidal gold, phenyl boronic acid PEG-succinate, streptomycin, lincomycin DNA/DNase digest, glycogen/starch amylase digest (50/50), benzidine Proteinase K/trypsin casein digest (50/50), theophylline, puromycin nucleotide pnpa-gln 10% Tacsimate Control

Experiment Va samarium(III) chloride hexahydrate 163 Calcium chloride dihydrate, magnesium chloride hexahydrate, manganese (II) chloride tetrahydrate, zinc chloride, urea 166 Nickel(II) chloride hexahydrate, copper(II) chloride dihydrate, Cobalt chloride, molybdenum(III) chloride, cadmium chloride hydrate 169 Control 172 Sulfanilic acid, 1,3,5-pentanetricarboxylic acid, 5-Sulfoisophthalic acid monosodium salt 175 Naphthalene-1,3,6-trisulfonic acid trisodium salt, 3,5-Dinitrosalicylic acid, trans-1,2-cyclohexane-dicarboxylic acid 178 Trimethylamine N-oxide, L-proline, L-glutamic acid, betaine monohydrate, taurine 181 Nicotinamide, cystamine dihydrochloride, 1,2-diaminocyclohexane sulfate 184 2,7-Naphthalenedisulfonic acid disodium salt, 1,5-Naphthlenedisulfonic acid disodium salt, sulfanilic acid 187 2,6-Naphthalenedisulfonic acid disodium salt, 4-aminobenzoic acid Naphthalene-1,3,6-trisulfonic acid trisodium salt 190 Control

a In experiment V the cocktails are numbered as sets of threes, with the first in each set at pH 5.8, the second at pH 6.8, and the third at pH 7.8. For example, cocktail 34 is at pH 5.8, 35 at 6.8 and 36 at 7.8.

X-ray diffraction data were recorded on an R-axis laboratory system using a Rigaku RU-200 rotating anode source with Osmic mirrors, or they were collected by remote Internet procedures on beam line 7-1 at SSRL. Data were processed and scaled using the program d*Trek.5 CNS6 was used for molecular replacement and refinement, though PHASER7,8 was also used for molecular replacement as well. Model fitting and rebuilding was carried out using the programs O9 and COOT,10 and ligand interactions with proteins were interpreted and illustrated with LIGPLOT.11 Figures were also made with the program PyMOL.12

Results and Discussion Experiment IV: Crystallization. Experiment IV had rather little to recommend it, no matter how one chose to score it, being generally a disappointment. Although some crystals took up strong color, there was nothing to suggest that the dyes, antibiotics, or drugs had any remarkable effect on the crystallization behavior of the macromolecule test set. There were no individual cocktails that stood out above the others, thus it would be pointless to present a scoring matrix or a histogram of successes for Experiment IV, as we did for the earlier experiments. There was, however, one, perhaps significant, finding that emerged from Experiment IV. From the earlier experiments came the realization that the crystallization of some proteins appeared to be promoted by mixtures containing oligosaccharides, oligonucleotides, and oligopeptides, this along with the observation that, with both PEG and salt precipitants, a mixture of the 20 amino acids naturally occurring in proteins also appeared to be an effective enhancer of crystallization. It occurred to us that digests of some macromolecules, such as casein, with proteases of various sorts, or glycogen and starch with amylases, might provide an array of hydrolytic products, i.e., mixtures of peptides or oligosac-

charides, that might serve as well as, or better than, defined mixtures made of individually selected molecules. We made a number of such digests from proteins, glycogen, starch, RNA and DNA, and included them in Experiment IV. Indeed, this idea appeared, from the results, to have some merit. If one gathered together all of the cocktails that produced what we might consider to be “exceptional results,” i.e., crystals for a certain protein where there were otherwise none, large, single crystals where there were otherwise only microcrystals or clusters, or where crystals of some unusual morphology had appeared, then these cocktails seemed to have a common characteristic. A large fraction were composed of peptides or other oligomers, created by enzymatic degradation of naturally occurring macromolecules such as casein or glycogen. Although exceptional results in Experiment IV were disappointingly few, about 50, 75% were produced by cocktails containing one or more of the digests. This is impressive, because only 19 of the 96 cocktails, or about 20%, contained digests of any sort. One digest that appeared in several successful trials, for example, was bactotryptone, a commercially available proteolytic digest of casein used in cell culture media. Experiment V: Crystallization. Experiment V had a great deal to recommend it, and was, in our opinion, one of the most effective experiments of our series. As shown by the scoring matrix in Figure 1, and the histogram of Figure 2, no one cocktail stood out as having uncommon efficacy with a wide range of proteins. As we pointed out in our previous paper2,13,14 and above, some of the most impressive indicators of a successful cocktail escape detection by such statistics. These occur when a specific protein crystallizes from only one, or only


Larson et al.

Figure 1. Scoring matrix for the 64 different cocktails, each formulated at 3 pHs, tested against 68 different proteins, viruses, and nucleic acids. The compositions of the cocktails are presented in Table 2, and the protein code that designates each macromolecule is given in Table 1. A dot signifies that the macromolecule crystallized, a diamond that the crystals represented an unusual occurrence. The phi indicates that crystals did not grow, and the square indicates that crystals were observed but may or may not be of the macromolecule. A question mark indicates that the presence of crystals was indeterminate.

a very few, cocktails, or when some exceptional result for an individual macromolecule is confined to only one or a few cocktails. Inspection of Figure 1 shows that there was an impressive number of cases of the appearance of “silver bullets,” where only one cocktail, or a very limited set of cocktails, produced crystals, or some exceptional result, when all others failed. Often, we subsequently found that when a small set of cocktails produced exceptional results, they contained a common component, or shared some obvious characteristic. It is worth examining some of these here in more detail. (1) Yeast invertase was noteworthy. Although we had included it in all of our previous screening experiments, it had

never crystallized. It persisted, generally, in yielding only phase separation. In this experiment, as in the others, it again failed to crystallize from any cocktail, with the exception of one. Invertase crystallized as thick clusters of needles in cocktail #106. Significantly, it crystallized from this cocktail at all three pHs, hence, in triplicate. Cocktail #106 includes O-phosphoserine/O-phosphotyrosine/6-phosphogluconic acid. The result was reproducible. (2) Yeast phenylalanine tRNA was among the nucleic acids included in our macromolecular test set. It gave generally clear drops in 95 of 96 samples, but crystallized at two of the three pHs, as shown in Figure 3(a), in the presence of cocktail #88.


Crystal Growth & Design, Vol. 8, No. 8, 2008 3043

Figure 2. A histogram showing crystallization patterns for the 64 independent cocktails used in Experiment V. Each cocktail was tested at three different pH values, but positive results at multiple pHs were treated as a single successful trial. The solid line denotes the number of macromolecules that crystallized for each cocktail. The compositions of the cocktails are found in Table 2.

Figure 3. Crystals of (a) yeast phenylalanine tRNA, (b) human hemoglobin, (c) pig pancreas R-amylase, (d) papain, (e) rabbit serum albumin, (f) thaumatin [tetragonal], (g) thaumatin [orthorhombic], (h) brome mosaic virus coat protein, (i) Escherichia coli leucine tRNA, (j) soy bean trypsin inhibitor, (k) bacterial amylase, (l) Candida lipase, (m and n) cow milk β-lactoglobulin, (o) sweet potato β-amylase.

This contains 2-aminobenzenesulfonic acid/2,6-naphthalene disulfonic acid/1,3-benzenedisulfonic acid. (3) Human hemoglobin yielded principally precipitates and phase separation in virtually every trial, save three. These were #73, #115 and #148. The crystals were large, rather thin plates of unusual morphology, as seen in Figure 3(b). Subsequent X-ray diffraction analysis of the crystals showed them to be of a curious and previously unreported unit cell. The crystals were of space group P6122 (or P6522) with a ) b ) 54 Å, and c ) 671 Å. The three cocktails that produced the crystals were composed of (#73) poly(3-hydroxybutyric acid/1,3,5-pentanetricarboxylic acid/trimesic acid, (#115) canavanine sulfate/

cystathionine/cystine, and (#148) 2-sulfobenzoic acid/5-sulfosalicylic acid/2′-deoxyuridine-5′-monophosphate. (4) Pig alpha amylase crystallized in three cocktails as thick needles, seen in Figure 3(c), of quite different morphology than we commonly observe.13,14 In cocktails #121 and #145, it crystallized in two of the three pHs, and in #158, one of the three. The first of the two cocktails was composed of oligopeptides, (#121) gly-gly/lys-lys-lys-lys-lys/asp-leu, (#145) leu-glygly/ala-ala/gly-gly. The third cocktail was a mixture of common organic buffers (#158) MES/PIPES/BICINE. (5) In the entire screen of 192 samples using the protein papain, there was only one trial that contained crystals, and we


were subsequently able to verify that they were indeed protein. These appeared in cocktail #148 which contained 4-sulfobenzoic acid/5-sulfosalicylic acid/2′-deoxyuridine-5′-monophosphate. The crystals are shown in Figure 3(d). In previous experiments2 we also found that the crystallization of papain was sometimes promoted by the inclusion of mononucleotides in the mother liquor. (6) Thaumatin also gave quite a remarkable result. Thaumatin is readily crystallized in the presence of tartrate, yielding large, well diffracting, tetragonal bipyramids15 like those in Figure 3(f). The crystallization screen here, however, lacked tartrate. We observed in cocktail #148 small spidery crystals, but whether they were protein could not be determined. No other sample contained any crystals except one, and that was in the presence of cocktail #68. These, as shown in Figure 3(g), were thick blades of rectangular cross section growing in a single cluster. We were able to reproduce the result by repeating the conditions in subsequent experiments. The crystals, upon analysis by X-ray diffraction, had an orthorhombic unit cell of space group P212121 and unit cell dimensions a ) 74.4 Å, b ) 53.3 Å, and c ) 52.3 Å. The crystals diffracted to 1.6 Å resolution. A structure determination from crystals having this same unit cell was previously reported,16 but to a resolution of only 3.2 Å. Cocktail #68 contained anthrone/N-(2-acetamido)-2-aminoethanesulfonic acid/Congo red. (7) There is no entry in the PDB for bovine erythrocyte ubiquitin, except in complex with other proteins, although we have crystallized it in some previous experiments. In Experiment V, virtually every drop was clear throughout the screen, with the exception of cocktail #177, which produced a few small and unimpressive crystal clusters. Cocktail #58, however, was thick with needle crystals, and this was true for all three of the pHs tested. Cocktail #177 contained naphthalene-1,3,6-trisulfonic acid/3,5-dinitrosalicylic acid/trans-1,2-cyclohexanedicarboxylic acid. Cocktail #58, which, as Figures 1 and 2 show, proved successful for a large number of proteins, contained 4-nitrobenzoic acid/2,5-pyridinedicarboxylic acid/mellitic acid. The latter compound, mellitic acid, has appeared frequently in successful trials throughout all of our experiments. (8) Like ubiquitin, there is no entry in the PDB for rabbit serum albumin. The albumin initially failed to crystallize in any trial of Experiment V except for one, and that was cocktail #101. That mixture yielded an impressive corona cluster of thick blades. Only after two additional months had passed did some crystals begin to appear in other drops, as found in the scoring matrix of Figure 1. Cocktail #101 contained phenyl urea/pcoumaric acid/sulfaguanidine. Subsequent X-ray diffraction analysis of the crystals showed them to have a primitive orthorhombic unit cell of space group P212121 with dimensions a ) 74.9 Å, b ) 81.0 Å, and c ) 105.2 Å. There were a substantial number of cases where small, unimpressive crystals, microcrystals, or crystals of questionable composition were observed throughout the screen, but where one or a few trials produced crystals that were exceptional in some sense. (9) A cleaved form of the coat protein of brome mosaic virus, in the absence of its genomic RNA, will reassemble into T ) 1 icosahedral particles.17 These particles were prepared, crystallized, and the structure of their tetragonal crystals solved by diffraction analysis some years ago.18 It is not clear, however, under what conditions the coat protein is assembled as icosahedral particles, or exists as individual protein units. The protein used in Experiment V, thus, carries this uncertainty. In Experiment V we observed microcrystals in six or more samples

Larson et al.

throughout the screen, but unusually large masses of needles in great abundance, seen in Figure 3(h) that were roughly an order of magnitude greater in size. These were observed in three cocktails, #58, #86, and #92. It was noteworthy that the exceptional crystals appeared in #58 at all three pHs tested, and at two pHs for each of the other cocktails. Thus there was good internal reproducibility. Cocktail #58, as noted above, and whose contents are given there, was among the most outstanding cocktails tested for many proteins, including this one. The other two cocktails are interesting for another reason. Cocktail #92 is composed of 5-sulfoisophthalic acid/cinnamic acid/1,4cyclohexanedicarboxylic acid, while #86 has the composition 2,7-naphthalene disulfonic acid/cinnamic acid/azelaic acid. The common compound, cinnamic acid, is suggestive. (10) Leucine tRNA was another of the nucleic acids that we included in the macromolecular test set for Experiment V. It was among the more significant of the RNAs because it had never previously been crystallized. In this experiment, microcrystals and generally small, misshapen plates were observed in nine of the trials. The tRNA had crystallized, but the crystals were unimpressive at best. In the presence of cocktail #166, however, the quite respectable clusters of plate crystals shown in Figure 3(i) were found. Cocktail #166 contains a mixture of divalent, transition metal ions, Ni++/Cu++/Co++/Mb++/Cd++. This result makes good sense, because we know from experience that such ions can dramatically affect the crystallization of nucleic acid molecules, and can be tightly bound between molecules in their crystals. (11) The results with soybean trypsin inhibitor were among the most impressive we obtained in some ways, though that would not be apparent from the scoring matrix of Figure 1. Most of the trials for this protein showed oiling out, precipitate, and in rare cases microcrystals or small needles. In one, and only one sample, however, there was a cluster of broad, thick, plates, and these are shown in Figure 3(j). When the protein was subsequently set up under identical conditions with the cocktail, the crystals were readily reproduced. Subsequent X-ray diffraction analysis showed the crystals to have a primitive monoclinic unit cell of dimensions a ) 45.9 Å, b ) 39.9 Å, c ) 55.9 Å, and β ) 100.6°. The cocktail that produced the results was a mixture of six amino acids, tyrosine/phenylalanine/tryptophan/ histidine/isoleucine/leucine. We found this noteworthy, because in our previous report2 we observed that mixtures of amino acids had a propensity to promote the crystallization of a wide range of proteins. (12) The lipase from Candida rugorosa gave microcrystals and small, misshapen crystals of plate and needle habit in many samples. In two cocktails, however, beautiful tetragonal bipyrimids, like those shown in Figure 3(l), were found, and these were #125 and #146. Both of these cocktails, like those for pig alpha amylase above, were composed of peptides, and one peptide in both mixtures was common to both proteins. #125 is composed of leu-gly-gly/gly-gly-gly/leu-gly and #146 of leugly-gly/ala-ala/gly-gly. (13) A result similar to that for the lipase was also obtained for the protein β-lactoglobulin from cow’s milk. Again there were fine needles and small disordered clusters of microcrystals in a great many of the trials, but tetragonal bypyramids in only one trial. These are shown in Figure 4(f). The cocktail was #37, which had only two components, 2,6-naphthalenedisulfonic acid/ 2,7-naphthalenedisulfonic acid. (14) Rabbit muscle lactate dehydrogenase has no entry in the PDB, though we have crystallized it in previous experiments. In Experiment V, it produced small, microneedle puffs and balls



Figure 4. Crystals of (a and b) pig heart citrate synthase, (c and d) bovine superoxide dismutase, (e) apotransferin, (f) cow milk alpha lactalbumin, (g and h) proteinase K, (i and j) rabbit muscle creatine kinase, (k) yeast hexokinase, (l) Bence-Jones KWR, (m) xylanase, (n) bovine ribonuclease A, and (o) bacterial glucose isomerase.

throughout the screen. In one cocktail, however, #145, perfect rectangular prisms and thick needles were observed, and this was true at two of the three pHs tested. The cocktail’s composition was, again, a mixture of peptides, leu-gly-gly/alaala/gly-gly. There are two particularly outstanding cases that deserve attention because they illustrate how the active ingredient of a cocktail can be identified with a fair degree of certainty. These involved the proteins citrate synthase from pig heart and lactoferrin from milk. (15) For citrate synthase, there were fine needle crystals and clusters for a moderate number of trials throughout the screen, as is evident from the scoring matrix in Figure 1. In four cocktails, needles were absent and in their place were large bipyramids and thick plates as seen in Figure 4(a) and (b) (space group C2221, a ) 104.3 Å, b ) 197.7 Å, c ) 50.63 Å). These were clearly exceptional. Subsequent experiments in which citrate synthetase was again set up with these four cocktails demonstrated that the crystals were readily reproduced. The cocktails were #3, #78, #153, and #183. The compositions of these four cocktails were (#3) anthrone/phenylglyoxal/cystamine, (#78) cystathionine/citruline/cystamine, (#153) cystamine/aspartame/benzamidine, (#183) nicotinamide/cystamine/1,2-diaminocyclohexane. It is difficult, from inspection of these compositions, not to conclude that cystamine has a powerful influence on the crystallization of citrate synthase. (16) For lactoferrin, small, undistinguished crystals or microcrystals were seen in three samples, but large, orthorhombic prisms of strikingly good morphology were seen for four cocktails, #4, #22, #82, and #175. More impressive still was that the same exceptional results were observed at all three of the pHs tested for each cocktail. Thus, the results were internally reproduced in triplicate. The compositions of the cocktails were illuminating. They were (#4) 3,5-dinitrosalicylic acid/2,5pyridinedicarboxylic acid/1,5-naphthalenedisulfonic acid, (#22) 3,5-dinitrosalicylic acid/aspartame/4-aminobenzoic acid/(#82) 5-sulfosalicylic acid/3-aminobenzenesulfonic acid/3,5-dinitrosalicylic acid, and (#175) naphthalene-1,3,6-trisulfonic acid/3,5dinitrosalicylic acid/1,2-cyclohexanedicarboxylic acid. Here, the conclusion seems inescapable that 3,5-dinitrosalicylic acid is the primary cause of the exceptional crystals. Of the eight nucleic acid molecules tested in the experiment, five of the tRNAs crystallized. Two of the tRNAs failed to crystallize, as did the STMV-RNA. The crystals that were

grown, with the exception noted above, were not of a size or quality for immediate X-ray diffraction analysis, but the results provided starting points for optimization. The results of Experiment V were analyzed to identify those cocktails that produced the greatest number of exceptional positive results among all the macromolecules, and those that produced the greatest negative results among those proteins that otherwise crystallized readily throughout the screen. These were simply extracted from the scoring matrix of Figure 1. There were 17 cocktails that produced three or more exceptional positive results, and 9 of these, 4 or more such results. There were also 12 cocktails which produced three or more exceptional negative results, and 10 of these produced 4 or more. There is a correlation between the two sets, and this is evident by inspection of the histogram in Figure 5. Seven cocktails, or 41% of the entries in one set, were in common with the other. If only the first tier entries are considered (i.e., those producing exceptional results greater than 4), the correlation is even higher, with 4/9, or 44% in common. The conclusion, which is entirely reasonable, would seem to be that specific cocktails, and their components, can have an effect on the crystallization of individual proteins, but that effect can be negative or positive, depending on the protein, and its interaction with the small molecules. Some of the compounds that appear on one list multiple times or on both lists are intriguing. Cystamine, which was determinant in the crystallization of citrate synthase (above) is on the positive list once, and on the negative list twice. 3,5-Dinitrosalicylic acid, which clearly dominated the crystallization of lactoferrin (above), also appears on both lists. Naphthalene-1,3,6-trisulfonic acid appeared twice in the positive set, and in the negative set as well, as did 1,2-diaminocyclohexane. 4-Nitrobenzoic acid appeared once in each set. It was interesting, and perhaps significant, that cocktails #121 and #145 were not only common to both sets, but the only peptide mixtures to appear in either set. Yet there were 11 peptide cocktails investigated overall. Thus, the commonality appears to be statistically meaningful. Less surprising is that cocktails #160, #163, and #166, all containing, or composed entirely of, positively charged metal ions, were common to the two sets. Indeed, we know from experience, and some focused research,19 that such ions can strongly affect macromolecular crystallization, in both a positive and a negative sense. The data shown here simply bears that out.


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Figure 5. In this histogram the upper bars represent the number of favorable “unusual occurrences” for each cocktail used in Experiment V, and the negative bars the number of times a cocktail appeared to inhibit the crystallization of a macromolecule with a high propensity to crystallize. A correlation between cocktails yielding the positive and negative results is evident.

Finally, we might note that the cocktail composed of a mixture of three common buffers, MES/PIPES/BICINE, appeared in both sets, positive and negative. This should, perhaps, not be surprising, as we know, again from past experience (see Table 1 of ref 2), that organic buffer ions such as these have frequently been found in crystal lattices by X-ray diffraction analysis. The data here serves further notice that the particular buffer used in crystallization experiments, and not just the pH it produces, may have more relevance to success or failure than we might have guessed. X-ray Diffraction Results. X-ray diffraction analyses were carried out on several dozen crystals grown in Experiments IV and V. In some cases, particularly where we felt the resolution was insufficient to reveal a small molecule clearly in difference Fourier maps, these were only preliminary analyses to obtain unit cell parameters. In other cases, data were collected to high resolution and difference Fourier maps computed. We present here some illustrative examples of the analyses. Shown in Table 3 are unit cell parameters and space group symmetries for the various crystals that we analyzed. Noteworthy among them are a number of unit cells that, to our knowledge, have not been previously reported. Included are primitive orthorhombic unit cells for rabbit serum albumin, concanavalin B, and one for porcine trypsin. New monoclinic unit cells were obtained for rabbit muscle creatine kinase, soybean trypsin inhibitor, and porcine trypsin. A new and unique hexagonal unit cell for human hemoglobin was observed. Multiple unit cells were found for the same protein grown in the presence of different cocktails, such as thaumatin, porcine and bovine trypsin, beef catalase, and fungal lipase.

In general, we placed trust in difference Fourier maps only when they extended to resolutions beyond 2 Å, and when the refined structures on which they were based had low R factors and good geometry. Because so many of the cocktail components were of small molecules of low electron density, had no major features such as aromatic rings, and often presented opportunity for disorder, we were not surprised to find that no ligand was apparent in many cases. We addressed that issue in some detail in an earlier paper.1 Presented here are synopses of several examples where ligands were successfully found in crystals from Experiments IV and V. They are necessarily brief and crystallographically incomplete, but they will be described in greater detail elsewhere. (1) Figure 6 illustrates results obtained from Fo - Fc difference Fourier maps at 1.7 Å resolution for ribonuclease A grown in the presence of a cocktail (Experiment IV, #80) containing tobramycin/UMP/basic fuchsin. The crystallographic unit cell has space group P21, with a ) 30.7 Å, b ) 74.9 Å, c ) 50.5 A, β ) 107.8°. The asymmetric unit contains two molecules of RNase A, and the difference Fourier map reveals two masses of electron density, having similar shapes, associated with each. The masses of density occupy portions of the active site clefts, the pyrimidine binding sites, but form partial interfaces with other protein molecules in the unit cell as well. The densities can readily be fitted by UMP, but not with any other component of the small molecule cocktail. As illustrated in the diagrams of Figures 6(a) and 6(b), both of the UMP molecules form extensive interactions with two different protein molecules within the lattice, and bind together the RNase A throughout


Crystal Growth & Design, Vol. 8, No. 8, 2008 3047 Table 3

protein soybean trypsin inhibitor rabbit albumin thaumatin bovine RNase A proteinase K lysozyme rabbit creatine kinase pig citrate synthase xylanase lipase bovine trypsin

pig trypsin

R-lactalbumin superoxide dismutase human hemoglobin concanavalin B taka amylase bovine catalase canavalin glyceraldehyde 3-PO4dehydrogenase glucose isomerase concanavalin A horse hemoglobin pig insulin sweet potato β-amylase β-lactoglobulin Bacillus alpha amylase

a (Å)

b (Å)

c (Å)

β (deg)

symmetry

resolution (Å)

previously obsd

45.9 74.9 74.4 58.6 30.7 55.1 101.8 67.7 78.1 133 259 198 76.3 40.2 139.3 54.3 62.5 138.4 55 46.4 58.5 71.0 60.7 113.2 104.3 54.0 48.0 50.5 77.0 66.4 89 173 137 137 82.4 94.0 81.2 68.5 106.7 62.3 62.1 80.9 128.2 62.0 91.7

40.8 81.0 53.3 58.6 74.9 55.1 33.3 67.7 78.1 69 68 198 76.3 38.6 139.3 58.4 64.2 138.4 55 53.5 58.5 50.8 103.5 113.2 197.7 54.0 61.9 67.3 91.7 102.5 140 173 137 150 99.2 99.4 91.1 115.4 62.3 62.3 79.9 80.9 128.2 62.0 150.0

54.8 105.2 52.3 151.8 50.5 39.2 74.0 103.0 37.4 237 133 71.3 198.2 56.9 80.5 66.3 69.7 150.5 109 75.8 137.3 125.5 115.5 107.2 50.6 671.50 130.2 130.6 150.0 75.0 231 237 76 133 186.5 103.0 99.7 121.8 54.5 130.1 107.9 33.4 65.9 181.0 77.0

99.2

P2 or P21 P212121 P212121 P41212 P21 P31 C2 P43212 P43212 P2 or P21 C2 I422 P41212 P21 P61 P212121 P212121 P3121 P3121 P212121 P412121 P21 P212121 R32 C2221 P6122 P212121 P212121 P212121 P21 P212121 P3121 R3 C2221 P212121 I222 P212121 P212121 C2 P31 P212121 R3 P42212 P4(1)22 P212121

2.8 3.4 1.6 1.2 1.7 1.3 2.1 1.1 1.1 4.5 4.0 4.0 1.7 1.1 2.2 1.2 1.6 2.2 1.0 1.2 1.4 1.3 1.7 2.7 2.4 4.8 1.9 1.5 2.3 2.1 2.6 2.5 2.4 2.6 2.0 2.0 2.1 2.5 1.7 1.7 2.1 2.0 3.0 4.5 1.6

no no yes yes yes no yes yes yes no no yes yes yes yes yes yes no yes yes no no no no yes no no yes no yes yes yes yes yes yes yes yes yes yes no yes yes yes no no

the crystals. The intermolecular interactions are undoubtedly responsible for the appearance of the crystals. (2) Lysozyme, grown in its familiar tetragonal unit cell (space group P43212, a ) b ) 78.1 Å, c ) 37.5 Å) was obtained in the presence of a cocktail (Experiment V, #43) containing 3,5-dinitrosalicylic acid/4-aminosalicylate/salicylamide. A ligand was clearly present in the crystal lattice and 4-aminosalicylate superimposed on the Fo - Fc density with good fidelity. The hydrogen bonds formed between the small molecule and the two protein molecules that it links are shown in Figure 7(a). In earlier experiments, we analyzed and described another lysozyme-ligand complex in which the ligand was p-amino benzoic acid. That complex was similar to this one with 4-aminosalicylate, but clearly not identical, as the two ligands failed to even approximately superimpose. The lysozyme from Experiment V, #43, exhibited no electron density at its active site, only the interfacial 4-aminosalicylate. We carried out similar X-ray analyses of three other lysozyme crystals grown in Experiment IV in the presence of cocktails #82, #78, and #34. The Fo - Fc difference Fourier maps showed, in all three cases, the identical difference density in the active site cleft. Because the three cocktails contained no common compounds, they provided

107.8 90.2 97.4 97.4 114.13 110.6

99.5

104.1

110.1

no explanation. The difference density, however, could be fitted quite well to a molecule of HEPES, the buffer that was used throughout the experiment, and was present in all of the cocktails. (3) Proteinase K was crystallized in its common tetragonal unit cell (space group P43212, a ) b ) 67.7 Å, c ) 103.0 Å) in the presence of 5,5-diphenylhydantoin/digalacturonic acid/ inosine monophosphate. The only difference density lay exactly on a crystallographic 2-fold axis, and it clearly suggested the ring of one of the small molecules lying on the axis. Neither 5,5-diphenylhydantoin nor inosine monophosphate provided any convincing fit to the density, even if disorder were allowed. One sugar component of the digalacturonic acid, assuming it to be 2-fold disordered, fitted the density quite well since it virtually superimposed upon itself even after rotation. Inspection of the maps at lower contour levels revealed the two, halfoccupied, dyad related alternate positions for the second sugar ring. The intermolecular interactions made by the digalacturonic acid and protein molecules within the crystal lattice are illustrated in Figure 7(b). (4) Porcine trypsin was crystallized in a previously unreported unit cell (space group P21, a ) 71.0 Å, b ) 50.8 Å, c ) 125.5 Å, β ) 99.5°) in the presence of benzamidine/pyromellitic acid/ mellitic acid, and the data collected to 1.3 Å resolution. As in


Larson et al.

Figure 6. LIGPLOT diagrams illustrating the interactions of the two uridine mononucleotides with the two RNAase A molecules comprising the asymmetric unit in the monoclinic complex crystals.

Figure 7. In (a) is a LIGPLOT diagram showing the intermolecular interactions of 4-aminosalicylate with two lysozyme molecules. The small molecule resides within a gap normally filled by water molecules that separates two protein molecules in the tetragonal crystal lattice. In (b) a similar diagram shows the interactions made by a galacturonic acid molecule lying between 2-fold related proteinase K molecules in the lattice of its tetragonal unit cell.

previous studies,1,4 benzamidine was found in the active site of all molecules in the crystal. In this crystal, however, there were four protein molecules in the asymmetric unit. In addition, two additional benzamidines were found at the interfaces of the

protein molecules, and nearby each, a pyromellitic acid molecule. These latter small molecules lay at the junctures of three trypsin molecules. The intermolecular, lattice interactions of these small molecules are presented in Figure 8. The benzami-



Figure 8. There are two pyromellitic acid and two non-active site bound benzamidine molecules per asymmetric unit in these unique crystals of porcine trypsin. In (a) and (b) are shown the LIGPLOT diagrams of the interactions made by the two pyromellitic acid molecules, and in (c) and (d), the corresponding illustrations for the two benzamidine molecules.

dine and the pyromellitic acid molecules bind together the protein molecules of the asymmetric unit through patterns of hydrogen bonds, again promoting the formation of lattice contacts. (5) Lipase from Candida was crystallized in Experiment IV #66 using a cocktail of o-nitrophenylgalactoside/adenosine monophosphate/quinaldine red (space group P61, a ) b ) 139.3 Å, c ) 80.5 Å). Two independent masses of electron density appeared in the difference Fourier map, and they could readily be fitted by models of AMP, a component of the cocktail in which they were grown. The nucleotides, superimposed on their corresponding difference electron density, are shown in Figure

9. Neither the dye nor ONPG, the other small molecules present, could be persuasively fitted to the density. In both cases, the nucleotides were sandwiched between two protein molecules in the crystal lattice, where they made extensive interactions with both. (6) We have commonly grown crystals of thaumatin in its familiar tetragonal unit cell15 in numerous experiments (space group P41212, a ) b ) 57.6 Å, c ) 149.7 Å). The crystals are readily recognizable by their characteristic tetragonal bipyramidal habit. In experiment V #67, however, crystals appeared which had a distinctly different morphology. The cocktail of #67 contained anthrone/Congo red/N-(2-acetamido)-2-amino-


Larson et al.

Figure 9. In (a) and (b) are seen the two independent molecules of AMP that fill voids in the crystal lattice of Candida lipase, as they are superimposed on their corresponding difference electron density.

Figure 10. In (a) the conformation of the carboxy terminus in the presence of N-(2-acetamido)-2-aminoethanesulfonic acid is shown superimposed on its disposition in the absence of ligand. The difference electron density belonging to the conformationally altered carboxy terminus is superimposed. In (b) the density belonging to half a cystamine covalently bonded to the sulfur of cysteine 184 of pig heart citrate synthase is shown superimposed on its corresponding model. Interactions made with a second protein molecule in the crystal lattice, some through water molecules, are indicated.

ethanesulfonic acid. Data were collected on the crystals, which had no color, to 1.6 Å resolution. The crystals had a P212121 orthorhombic unit cell (a ) 74.4 Å, b ) 53.3 Å, c ) 52.3 Å). There was an entry (1THI) in the PDB from 198916 corresponding to this unit cell, but it consisted of only R carbon coordinates, and the resolution was reported to be only 3.8 Å. Using the model taken from the tetragonal unit cell, the orthorhombic crystals were solved by molecular replacement and refined. What emerged was the following. There was a single, somewhat elongated, mass of density deep within a cleft that divides the protein molecule, but which is also immediately juxtaposed with the carboxy terminus of a second thaumatin molecule, thereby forming a protein-protein interface. The difference density was inconsistent with either anthrone or Congo red. The sulfonic acid group and a portion of the remainder of a molecule of N-(2-acetamido)-2-aminoethanesulfonic acid could, however, be placed in the density. In the resultant structure, which will be described in more detail elsewhere, the last two amino acids at the carboxy terminus of one protein molecule completely reorient with regard to the original model in such a way that they interact directly through

the N-(2-acetamido)-2-aminoethanesulfonic acid with the other. Thus the small molecule here, accompanied by a minor conformational change, becomes the linchpin of the assembly. The conformational change involving the carboxy terminus superimposed on its difference electron density is shown in Figure 10(a). (7) As described above, one of the most convincing examples of a small molecule affecting the crystallization of a specific protein was that of cystamine with pig heart citrate synthase. Although it yielded crystals of a known unit cell, it allowed us to extend the resolution of the structure determination from 3.2 Å to 1.6 Å. Analysis of this high-resolution model showed a number of small, but nonetheless significant, conformational alterations when compared with the original model. At only one location, however, was there additional new density indicative of the presence of a small molecule. What we found was a distinctive, prominent density mass, seen in Figure 10(b), that was contiguous with the thiol group of cysteine 184. The elongated density extended away from the thiol toward a second protein molecule in the crystal lattice. Our interpretation, consistent with the chemistry of cystamine,


and the process of disulfide exchange, is that half of a cystamine molecule formed a disulfide bond with cysteine 184 on the surface of the protein. The amino portion of the half cystamine then proceeded to form a network of intermolecular interactions, involving both direct hydrogen bonds and bonds mediated by waters that stabilized the crystal lattice. Discussion The most significant result to emerge from these experiments is that they further support the hypothesis that small molecules can, in many cases, promote the crystallization of macromolecules by the formation of important lattice contacts, by linking protein molecules together in the crystal. In other cases, it is seen that new, previously unreported crystallographic unit cells were obtained as a consequence of the presence of the small molecules. Thus we maintain that the screening of small molecule libraries under a few select crystallization conditions provides a novel and often effective approach to macromolecular crystallization. It assuredly serves as a complement to methods currently in use. The X-ray diffraction results further provide a physicalchemical basis for understanding how the small molecules bind in the lattice, and how they serve to bring the protein molecules together, principally through the formation of networks of hydrogen bonds and electrostatic interactions. In all of the examples described above, even in spite of disorder in some cases, the small molecules were fulfilling the role that we originally envisioned. They were incorporated into the crystal lattices, formed intermolecular linkages between protein molecules, stabilized protein-protein interfaces, or created them, and in several ways promoted crystal growth. These sorts of results, however, characterize less than half of all of the systems that we fully analyzed by X-ray diffraction analysis. For many of the protein-small molecule combinations that we studied, even when it was evident from reproducible experiments that a small molecule was essential in order to grow crystals of a particular protein, we could observe no electron density in difference Fourier maps that we could ascribe to any component of the corresponding cocktail. Small molecules were necessarily present in the mother liquor to promote crystallization, but they could not be found in the refined structure. We continue to be perplexed by these results, though they are little different from reports found throughout the literature regarding many macromolecular crystals. There are, of course, technical reasons why the small molecules might not appear in difference Fourier maps. The resolution of the data may be insufficient, the data of marginal quality, the small molecules insufficiently electron dense, etc. We dealt with some of these issues previously, but these explanations, it seems to us, are not sufficient to explain all of the cases where small molecules are not observed in a crystal. The question persists. We have considered this apparent paradox, and though we have reached no definitive conclusion, we have compiled several possibilities that seem to warrant further reflection. (1) There is some evidence20 that protein molecules are not necessarily held together in a crystal by direct protein-protein interactions, even those mediated by a water molecule, but by chains of water molecules. That is, clusters or strings of water molecules form networks of hydrogen bonds in the crystal that tend to “freeze” the macromolecules in their places. This effect has occasionally been visualized in high-resolution structure determinations. It may be that the small molecules that fail to appear in the crystals are in fact there, but disordered. Even so,


they could disrupt or change the water structure around and between macromolecules. Because the relationship between solute and solvent is so crucial in crystallization, this may be sufficient to explain the puzzling results. (2) The small molecules may be present in the crystal, but simply not ordered. They may be bound in a disordered manner to the protein molecules, they may be present in the bulk, unstructured solvent between molecules, in cracks and crevices and lumens. They may even be sequestered in gross crystal defects such as absences and at planar defects.21 It is difficult to see, however, why the small molecules in this case would be essential for, promote, or alter crystal growth, but it is possible. (3) Our findings tend to suggest that the small molecules may exert their most profound effect at the nucleation stage of crystallization. In particular, we found new crystal forms that previously were unobserved, and frequently obtained polymorphs as a function of the small molecules present. The unit cell of the crystal is almost certainly established within the critical nucleus, and the small molecules may work by enhancing, disrupting, or altering the pattern of clustering there. The small molecules may even be incorporated into the nucleus, thereby setting the pattern, but then become unnecessary for the propagation of the lattice once the unit cell is set. That is, the nucleus of the crystal may have a fundamentally different composition than the bulk of the crystal. This scenario may appear imaginative, but it was suggested twelve years ago by Rosenberger and colleagues22 to explain certain observations of apparent inhomogeneities in lysozyme crystals. Indeed there are other data, for example from X-ray tomography,23 that support the idea that the initial nucleus is physically or chemically different than the remainder of the crystal. (4) The small molecules may, in fact, all be incorporated into the growing crystals and promote crystallization by a common mechanism, the one we originally proposed of developing stable lattice interactions, but be weakly bound and lost from the crystal over time or due to temperature change. This would explain why they fail to appear in difference Fourier maps, but aside from the rigors of crystal mounting and data collection, it is difficult to understand why they would subsequently be lost after growth. (5) The small molecules may be transiently bound in solution to the protein molecules but be sacrificed to more favorable protein-protein interactions as the macromolecules enter into the crystal lattice. By binding to the macromolecules in solution they might stabilize them conformationally, serve as intermediates in the transition from fully solvated to partial solvation in the crystal, they might perturb in a favorable way the interactions between macromolecules, or alter the pattern of aggregation to encourage formation of critical nuclei. This may be testable by experiment, as alteration of these precrystalline interactions could affect the second virial coefficient of the mother liquor.24–26 Were this the case, then the effect of the small molecules might become apparent in static light scattering experiments. Whether the small molecules used in the screening for crystallization of macromolecules appear in the final structure or not is of minor consequence in general. The objective is to obtain the structure of the protein or nucleic acid, with or without ligands bound. Nonetheless, direct observation of the lattice interactions resulting from the presence of the small molecules continues to be instructive. We are convinced that further acquisition of the kinds of data that we present here


will ultimately lead us to an understanding of the relevant mechanisms. More importantly, perhaps, they will allow us to better define the classes of small molecules that will best serve our interests. The results of the experiments also provide evidence that the approach we have pursued in this work is effective for the crystallization of RNA molecules as well as for proteins and viruses. We conclude that it is likely to be useful when applied to virtually all biological macromolecules. Additional information regarding sources of chemicals, their formulation into cocktails, and their availability to the crystal growth community can be obtained from Hampton Research, Aliso Viejo, CA. E-mail: [email protected] or investigate www.hamptonresearch.com. Acknowledgment. The authors wish to thank Mr. Aaron Greenwood for preparation of the figures. This work was supported by NIH Grant GM074899 for the establishment of the Center for High Throughput Structural Biology.

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Larson et al. (8) Storoni, L. C.; McCoy, A. J.; Read, R. J. Likelihood-enhanced fast rotation functions. Acta Crystallogr., D: Biol. Crystallogr. 2004, 60 (Part 3), 432–438. (9) Jones, A. T. A graphics model building and refinement system for macromolecules. J. Appl. Crystallogr. 1978, 11, 268–272. (10) Emsley, P.; Cowtan, K. COOT model - building tools for molecular graphics. Acta Crystallogr. 2004, D60, 2126–2132. (11) Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 1995, 8 (2), 127–134. (12) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific, Palo Alto, CA, 2002. (13) Larson, S. B.; Greenwood, A.; Cascio, D.; Day, J.; McPherson, A. Refined molecular structure of pig pancreatic alpha-amylase at 2.1 Å resolution. J. Mol. Biol. 1994, 235 (5), 1560–1584. (14) McPherson, A.; Rich, A. X-ray crystallographic analysis of swine pancreas -amylase. Biochim. Biophys. Acta 1972, 285 (2), 493–497. (15) Ko, T. P.; Day, J.; Greenwood, A.; McPherson, A. Structures of three crystal forms of the sweet protein thaumatin. Acta Crystallogr., D: Biol. Crystallogr. 1994, 50 (Part 6), 813–825. (16) Kim, S. H.; de Vos, A.; Ogata, C. Crystal structures of two intensely sweet proteins. Trends Biochem. Sci. 1988, 13 (1), 13–15. (17) Lucas, R. W.; Kuznetsov, Y. G.; Larson, S. B.; McPherson, A. Crystallization of Brome Mosaic Virus (BMV) and T)1 Brome Mosaic Virus Particles following a structural transition. Virology 2001, 286, 290–303. (18) Larson, S. B.; Lucas, R. W.; McPherson, A. Crystallographic structure of the T)1 particle of brome mosaic virus. J. Mol. Biol. 2005, 346 (3), 815–831. (19) Trakhanov, S.; Quiocho, F. A. Influence of divalent cations in protein crystallization. Protein Sci. 1995, 4 (9), 1914–1919. (20) Frey, M. Water structure associated with proteins and its role in crystallization. Acta Crystallogr. 1994, D50, 663–666. (21) Malkin, A. J.; Kuznetsov, Y. G.; McPherson, A. Defect Structure of Macromolecular Crystals. J. Struct. Biol. 1996, 117, 124–137. (22) Rosenberger, F.; Vekilov, P. G.; Muschol, M.; Thomas, B. R. Nucleation and crystallization of globular proteins - what we know and what is missing. J. Cryst. Growth 1996, 168, 1–27. (23) Higuchi, Y.; Okamoto, T.; Yasuoka, N. The heterogeneity in a protein crystal revealed by synchrotron radiation. J. Cryst. Growth 1996, 168, 99–105. (24) George, A.; Wilson, W. W. Predicting protein crystallization from a dilute solution property. Acta Crystallogr. 1994, D50, 361–365. (25) Guo, B.; Kao, S.; McDonald, H.; Asanov, A.; Combs, L. L.; Wilson, W. W. Correlation of second virial coefficient and solubilities useful in protein crystal growth. J. Cryst. Growth 1999, 196, 424–433. (26) Wilson, W. W. Light scattering as a diagnostic for protein crystal growth-a practical approach. J. Struct. Biol. 2003, 142 (1), 56–65.

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