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The Role of Small Molecule Additives and Chemical Modification in Protein Crystallization Published as part of the Crystal Growth & Design virtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13). A. McPherson,*,† C. Nguyen,‡ R. Cudney,‡ and S. B. Larson† † ‡
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697-3900, United States Hampton Research, 34 Journey, Aliso Viejo, California 92656-3317, United States ABSTRACT: An alternative approach to promoting the crystallization of proteins is to enhance intermolecular contacts between macromolecules or to eliminate intermolecular interactions or interactions with solvent that might inhibit crystallization. Site-specific mutations have been employed, as have truncations by genetic or proteolytic means. There are, however, significant problems. Because the structure of the target macromolecule is unknown, there may be no good basis for the design of mutants or truncations. In addition, the approach requires that the protein be produced by recombinant DNA technology, which is frequently not the case. We have attempted to address these issues by initiating experiments based on two ideas. The first is that a wide variety of conventional small molecules might be systematically introduced into mother liquors during crystallization screening. By incorporation into the crystal lattice, the additional intermolecular interactions that the small molecules provide might enhance crystal nucleation and growth. A second approach that we are pursuing is the chemical modification of various amino acid side chains using traditional protein chemistry. We believe that in some cases chemically modified proteins might be induced to crystallize or crystallize better than the native protein.
’ INTRODUCTION A clear dependence of structural biology on the crystallization of proteins, nucleic acids, viruses, and other macromolecular complexes has developed, because the vast majority of molecular models are now derived from X-ray crystallography. Crystal growth has come to play a crucial role in the entire enterprise of structure determination both in the laboratories of individual investigators and in large structural genomics centers. The dependence is particularly acute with regard to the more intractable macromolecules such as membrane proteins and glycoproteins and to intricate complexes of macromolecules. Current approaches to macromolecular crystallization, while successful in perhaps 40% of cases, have not proven themselves able to successfully address many of the most biologically and medically significant problems. ’ INCLUSION OF SMALL MOLECULES IN THE MOTHER LIQUOR We have developed and are continuing to refine an alternative strategy to current protein crystallization methods based not on optimization of traditional variables such as precipitant concentration and pH but on the idea of identifying conventional and biologically active small molecules that promote crystallization through formation of favorable lattice contacts.1,2 Such small r 2011 American Chemical Society
molecules, traditionally referred to as “additives”, have often proven crucial to macromolecular crystallization. The strategy we have been exploring carries that idea to the forefront. The small molecule reagents, and their combinations, become the primary factor in the crystallization process. A review of the crystallization literature suggests that there are seven broad categories into which all of the small molecule additives can be placed. They are as follows: 1. Physiologically or biochemically relevant small molecules, such as coenzymes, substrate analogues, inhibitors, metal ions, or prosthetic groups. These bind at the active sites of enzymes or at specific sites elsewhere on protein molecules and may promote more stable, homogeneous conformations, or they may induce conformational changes into alternate states. Some early examples of the many now available are hemoglobin,3 lactate dehydrogenase,4 and dihydrofolate reductase.5 In any case, the ultimate protein ligand complex may exhibit a more monodisperse, less dynamic character. The pertinent molecules here are specific to the individual protein under study, and their selection for inclusion in mother liquors is amenable to Received: October 5, 2010 Revised: March 6, 2011 Published: March 16, 2011 1469
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2.
3.
4.
5.
6.
7.
rational analysis informed by the enzymology and biochemistry of the protein under study. That is, one considers all of the possible ligands of the protein and includes them in the screen of potential crystallization conditions. Chemical protectants. These include reductants such as BME, DTT, heavy metal ion scavengers such as EDTA and EGTA, and compounds intended to prevent microbial infection such as sodium azide, phenol, or chlorobutanol. These too are generally included for well-understood reasons, their effects are predictable, and their impact on the crystallization process is usually (but not always) of marginal significance. Solubilizing agents and detergents. These include quaternary ammonium salts,6 sulfobetains,7 chaotropes like urea,8 and a range of surfactant and detergent molecules.911 Because of the interest in membrane proteins, this class of additives has received extensive study and has been broadly applied to many proteins, including soluble proteins. Remarkably, there is still no consensus on which are most useful, which should be included in screening conditions, or even how they function in the solubilization of macromolecules. Poisons, as they have traditionally been called,12,13 were originally employed to reduce twinning. These are generally low concentrations, 15% w/v, of common organic solvents. They include compounds such as ethanol, DMSO, acetone, dioxane, butanol, or MPD. Their role in the crystallization process, even after 50 years of use, remains obscure. They likely enhance the solubility of proteins and slightly reduce the degree of supersaturation in the mother liquor, as well as lower the dielectric constant of the medium, but they may have other effects as well. Osmolytes, cosolvents, and cosmotropes are compounds that exert their effects at relatively high concentrations, 1 M or more, and include a wide range of molecules that include sucrose and other sugars, proline, TMAO, glycine, betaine, taurine, sarcosine, and a host of others.8,14 The effect of their inclusion in the mother liquor is to stabilize (or destabilize) the native conformation of the protein by altering the interaction of the protein’s surface with water or by altering the hydration layer and possibly the structured waters. It has been proposed that the conformations of proteins might be stabilized and their dynamic character might be reduced by providing the proteins with small molecules that could reversibly cross-link charged groups (carboxyl and amino groups) on the protein’s surface or form intramolecular hydrogen bonding networks using surface polar groups.15 The compounds that have been explored are usually multivalent such as diamino or dicarboxylic acid containing molecules or aliphatic moieties of various lengths carrying some combination of charged groups. It is not known whether the stabilization of proteins by this means is significant enough to affect their crystallization. The class of compounds useful for stabilizing proteins through noncovalent intramolecular bonds, as described above, may also help create and stabilize protein crystals by interposing themselves between protein molecules and forming intermolecular cross-links.13 These cross bridges may involve purely electrostatic interactions, or they may rely on hydrogen bonding arrangements as well. The compounds most favorable for forming such “lattice interactions” are,
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again, likely to be multivalent, charged compounds, but we might expect that their length, or “reach” would need be greater, since they would have to extend from one protein molecule to another. Some examples of these actually observed in protein crystals are the following: (a) Cd2þ in horse spleen ferritin16,17 (b) DNA oligomers in RNase A18 (c) Oligosaccharides in R-amylase19 (d) Tartrate in thaumatin20 (e) SO 4, PO4 , and other multivalent anions (many examples) (f) MPD and PEG (many examples) (g) Glutathione, sarcosine, HEPES, glycerol (many examples) (h) Short peptides in proteases A final class of additives not included above would be those materials or compounds that somehow serve to enhance nucleation, including unique surfaces. Pertinent here are low concentrations of PEG11,18 or other polymeric substances such as Jeffamine emulsified in solutions of high salt concentration.21,22 The microdroplets of the polymeric phase serve to concentrate the protein locally and provide an interface for nucleation to occur. This category should probably also include things like the gel used in cubic lipidic phase crystallization23,24 and surfaces that promote epitaxy and heterogeneous nucleation.25,26 Our fundamental hypothesis is (1) that conventional molecules having unique features may be bound by biological macromolecules, which may then be stabilized or induced to assume a more favorable conformation for crystallization, (2) that the small molecules may alter the interactions between macromolecules and their solvent and induce ordered association, or (3) that the small molecules may form reversible, crosslinks in a crystal lattice through hydrogen bonding, electrostatic, and possibly hydrophobic interactions and thereby promote formation of a crystal lattice. While it might appear that identifying specific molecules that promote the crystallization of a particular protein is an intractable task, there being an impossibly vast number of chemical compounds, this is not, in fact, the case. As we have shown in preliminary experiments,1,2 we are not obliged to evaluate compounds individually but can do so in clusters of various sizes. By grouping of compounds into formulations, sample matrices can be devised to test 200300 chemicals in a single 96 well screen. The only chemicals that need be considered are those of significant solubility in water that do not denature proteins. Further, available evidence suggests that the most suitable compounds are those bearing groups that engage in electrostatic or hydrogen-bonding interactions with proteins. Some time and effort will be required to assemble a sufficiently broad base of experience and refine reagent combinations, but eventually compounds offering the greatest potential benefit will emerge. While any one compound or reagent mix may have only a small chance of promoting the crystallization of a specific protein, the probabilities of success contributed by each reagent mix in a large set are additive. The problem we are currently addressing is to identify those molecules and compounds that can serve, for at least some proteins, to occasionally enhance the probability of a successful outcome. We have described elsewhere the results of experiments1,2 intended as initial steps in identifying classes of molecules and individual compounds that might be generally useful in promoting 1470
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Crystal Growth & Design the crystallization of macromolecules or that might have utility in increasing the probability that a specific protein crystallizes. The techniques, methods, and materials that we used in those and subsequent experiments are detailed there. In several experiments, we investigated the effects of various reagent mixes, generally having two to eight components, on the crystallization success of more than 80 different proteins. In these experiments, the compounds were not principally bioactive but were chemical compounds that might affect the solubilities of the proteins, their stability, their surface properties, or their interactions within a crystal lattice. In another experiment,1 bioactive and physiologically relevant compounds were tested on 66 proteins with the objective of finding combinations that might crystallize more readily or crystallize in a different form than the unliganded protein. In all of our experiments, more than 500 compounds were explored as possible additives. Only two fundamental crystallization conditions were used. One of these was based on 30% PEG 3350, the other on 50% TACSIMATE, both at pH 7. The experiments encompassed nearly 50 000 individual crystallization trials, using sitting drops, deployed both manually and robotically. Statistical analysis revealed a number of interesting clues that suggested those types of compounds most generally useful. Of particular significance was the finding that the use of reagent cocktails more than doubled the number of proteins that could be crystallized under the two basic conditions utilized, when compared with those two conditions free of any small molecules. The crystallization data provided persuasive evidence that, for many macromolecules, incorporation of one or more small molecules could be crucial to obtaining crystals. The results further indicated that certain classes of small molecules, such as dicarboxylic acids and diamino compounds of various sizes and geometries, promoted the crystallization of proteins in a general sense. In addition, different crystal polymorphs were produced of some proteins in the presence of various small molecules, and effects on the diffraction resolutions of some crystals were also observed. Results that complement ours were also reported based on independent experiments in other laboratories.27,28 To further test whether the underlying hypothesis was valid, that the small ligands did indeed serve to tether macromolecules to one another and thereby encourage lattice formation, X-ray analysis of at least a sampling of the crystals grown in the experiments was carried out. Only by this technique could detailed interactions within lattices be directly visualized, and the original idea rigorously evaluated. In subsequent papers,2932 we described analyses of crystalline proteins grown in the original experiments, each obtained in the presence of a “cocktail” of low molecular weight compounds. Another, independent analysis was carried out on bovine trypsin crystals grown in our experiments in the presence of benzamidine and protamine.33 In numerous cases, small molecules from different reagent mixes were bound and were clearly seen in difference Fourier maps. In those instances, we were able to unambiguously identify which component of the reagent mix the ligand represented. The cases investigated were most frequently those where the small molecule contained an aromatic ring, such as sulfanilic acid, or a larger ligand such as a nucleotide. X-ray diffraction analyses in which data were recorded from crystals grown in the presence of various reagent mixes and then used to calculate difference Fourier syntheses generally revealed the presence of ligands in the lattices at interfaces between protein molecules, consistent with the motivating hypothesis.
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In other experiments,30 on lysozyme and porcine trypsin crystals, p-amino benzoic acid and sulfanilic acid, respectively, were clearly observed to be present in the lattice and in both crystals served to establish an interface between protein molecules. This kind of binding was exactly what we might have anticipated. The same was true of trimesic acid in another lysozyme crystal, malonate, oxamic acid, and sorbitol in porcine trypsin crystals, and tartrate in crystals of thaumatin. In all of these cases, virtually every hydrogen bonding possibility inherent to the ligand was utilized in making the intermolecular interactions. On the other hand, some small molecules often do interact in more biochemically relevant ways with protein molecules and thus affect the way in which they crystallize. For example, in the case of benzamidine in trypsin, the ligand binds tightly at the active site, does not form intermolecular bonds, and yet enhances the crystallization of the protein. Without benzamidine present, under the same conditions, neither bovine nor porcine trypsin crystallizes. In this case, the positive effect of the small molecule must be exerted through stabilization of the protein structure or by its promotion of imperceptibly small alterations in conformation or disposition of surface groups. Of current emphasis are peptides produced by chemical synthesis, having lengths of two to five amino acids, and polypeptides derived by chemical and enzymatic digest of common proteins. Early reports from users of the SILVERBULLETS SCREEN (Hampton Research, Aliso Viejo, CA) indicated that these components of the kit appeared to be of particular effectiveness in crystallizing proteins. Our efforts are now directed toward defining which peptides are most useful in terms of (1) length, (2) composition, (3) charge distribution, (4) hydrophobicity/hydrophilicity, (5) geometrical properties, and ultimately (6) their sequence. We are currently exploring approximately 100 defined peptides, cocktails of the peptides, and protease digests of proteins that cover a spectrum of properties. We anticipate that these may provide guidance for future design.
’ CHEMICAL MODIFICATIONS OF PROTEINS It is becoming increasingly evident that one of the most powerful approaches for the crystallization of proteins is the direct modification of the protein itself. As Alan D’Arcy has pointed out, “your protein is your most important parameter”.34 Currently, one of the most profitable strategies is to purify a protein, screen it against 300500 crystallization conditions, and, if that fails to generate crystals of the protein, then make some genetic construct of the protein containing mutations of various kinds and repeat the screening process. Given the speed and reliability of current robotics systems, the screening for crystallization is both rapid and nonintensive in terms of investigator effort. The production of genetic constructs is not so, however. An additional approach is not to make point mutations in the protein but to make truncations either using proteases or genetically. Frequently these are accompanied by mass spectrometry analyses to precisely define what truncations are optimal, and occasionally it can be successful by amino acid sequence analysis designed to identify disordered regions. A problem with this approach is that making genetic constructs or truncations is time-consuming and often difficult, because each new construct must be purified for crystallization trials. In addition, in spite of considerable effort, it is still not clear what mutations are likely to be helpful in crystallization; thus 1471
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Table 1. Amino Acid Targets and Reagents amino acid
target
reagents 2-iodoacetamide, N-ethyl maleimide, bromoacetic acid, PCMB, Ellman’s reagent
cysteine
thiol
histidine
imidazole
diethyl pyrocarbonate, 2,3-butane dione, bromo phenacyl bromide
lysine
amino
acetic anhydride, formaldehyde, acetaldehyde
tyrosine
phenol
2-nitro-5-thiocyano benzoic acid, N-acetyl imidazole, KI/I, sodium periodate
arginine
guanidinium
phenyl glyoxal
methionine
sulfur
ozone, cis-platinate, hydrogen peroxide
cystine
disulfide
glutathione, DTT
aspartic acid glutamic acid
carboxyl carboxyl
carbodiimide carbodiimide
serine
hydroxyl
p-methyl sulfonyl fluoride
Table II. Some Difficulties with Chemical Modification • Cysteine and histidine often react with reagents intended for the other and for other amino acid side chains as well. • Lysine, glutamic acid, and aspartic acid are generally exposed on the surfaces of proteins but occur in relatively large numbers. • With the exception of cysteine and histidine, specificity is problematic. • Incomplete reaction of side chains may occur, leading to mixed products. • Some reactions may produce large local or even long-range conformational changes in specific proteins. • Reaction conditions must be compatible with the protein’s “comfort zone” of chemical and physical conditions. • Optimal reaction conditions may vary from protein to protein.
many constructs might have to be made and tested. Furthermore, this approach can only be applied when the protein has been cloned and expressed and is available for genetic manipulation. It would be far more efficient if the native protein obtained from the first round of cloning, expression, and purification could be modified in different ways that produced effects similar to genetic mutations. An alternative strategy is classical chemical modification of proteins using well-defined and widely used chemical reagents. Another way of stating this is that we use what 100 years of protein chemistry has taught us regarding the alteration of amino acid side groups and their effects on proteins’ physical chemistry. Indeed, the principal kinds of modifications, the reagents commonly used to achieve them, and the conditions under which they can be applied have been gathered together and detailed in Techniques of Protein Modification by Roger Lundblad.35 This book alone contains effective procedures for the modification of lysine, arginine, tyrosine, histidine, cysteine, methionine, and the carboxylic amino acids glutamate and aspartate. Furthermore, the modifications are not limited to one or a few but suggest, in some cases (lysine, glutamate, and aspartate), almost limitless possibilities. Encouragingly, most of the modifications can be carried out under physiological conditions that will not otherwise perturb proteins. We propose that chemically modified proteins, as alternatives to genetically modified proteins, might be used to effect crystallization when the native protein fails to crystallize. Outstanding examples of this approach is the methylation of lysine residues, first used by Rayment36 to effect the crystallization of the myosin S1 head, but subsequently used by several other investigators successfully with other proteins. The advantage is that the modifications can be carried out rapidly and with little effort and can be integrated into an automated crystallization screen. In a sense, once reaction conditions are refined, the reagents involved in modification can be considered simply as additives, as are the small molecules in the screening approaches described
above. No system for genetic manipulation is needed, and possibly, no extensive purification of the protein for each modification may be necessary. Table 1 lists those amino acid residues that are found in proteins, all of which are generally accessible on the surfaces and can be modified, each by a variety of chemical reagents. Table 1 also presents only some of the chemical modifications that have been used and proven to be effective. These are the reactions that we are addressing initially and will later expand upon. The residues involved in these modifications are almost exclusively those that are found on the surfaces of proteins. Thus they are commonly accessible, and they are residues that often participate in crystal lattice contacts and that form both salt bridges and hydrogen bonds between protein molecules in crystals. Interestingly, protein crystallographers acquired and utilized a substantial number of chemical modification approaches and technologies in the first 30 years of its existence. Multiple isomorphous replacements required the formation of heavy atom derivatives of crystalline proteins. While many of these took advantage of the chemical properties of the heavy atoms themselves and particularly their reactivity toward the thiol groups of cysteines and the imidizole groups of histidine, many did not. Though not comprehensive, many of these early applications were collected in the classic text by Blundell and Johnson.37 Although chemical modification offers an attractive approach to obtaining altered protein molecules, it is attended with significant problems, and these, of course, must be overcome. Most of these issues are presented in Table II, but chief among them are specificity and incomplete reaction products. Most proteins have several or even many residues of a particular type, each of which might serve as a target for a chemical reagent. This alone could produce a spectrum of products. Each of those targets, in turn, could yield multiple products due to incomplete reaction with the reagent. The problems, however, are not insurmountable, and we fully expect that in the future chemical modification will provide a parallel track for the enhancement of 1472
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Table III. Reagents in Experiment I 1. diethyl pyrocarbonate
13. N-acetyl imidazole
2. KI/I
14. platinum hexachloride
3. PMSF
15. subtilisin
4. sodium periodate
16. mercury acetate
5. Ellman’s reagent 6. Edmond’s reagent
17. 2-nitro-thiobenzoic acid 18. acetic anhydride
7. 2-iodoacetamide
19. hydrogen peroxide
8. bromoacetic acid
20. phenyl glyoxal
9. trypsin
21. 4-bromophenacyl bromide
10. PCMB
22. 2,3-butane dione
11. N-ethyl maleimide
23. diethyl carbodiimide
12. platinum tetrachloride
a protein’s crystallization potential. A set of reagents that is at present under investigation in our own laboratories is shown in Table III. Currently, we are applying the chemical modification approach to a range of proteins, most of which we can crystallize in their unmodified state. Initially we are using changes in the crystallographic properties (space group, unit cell dimensions, resolution, etc.) to evaluate which reagents might be most effective. Beyond that we will use the idea to address some now refractile problem proteins.
’ AUTHOR INFORMATION Corresponding Author
*Mailing address: University of California, Irvine, Dept. of Molecular Biology & Biochemistry, Room 560 Steinhaus Hall, Irvine, CA 92697-3900. Tel: (949) 824-1931. Fax: (949) 8248551. E-mail:
[email protected].
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(33) Viola, R.; Carman, P.; Walsh, J.; Miller, E.; Benning, M.; Frankel, D.; McPherson, A.; Cuillel, M.; Rupp, B. Operator-assisted harvesting of protein crystals using a universal micromanipulation robot. J. Appl. Crystallogr. 2007, 40, 539–545. (34) Dale, G. E.; Oefner, C.; D’Arcy, A. The protein as a variable in protein crystallization. J. Struct. Biol. 2003, 142 (1), 88–97. (35) Lundblad, R. Techniques in Protein Modification; CRC Press: Boca Raton, FL, 1995. (36) (a) Rayment, I. Reductive alkylation of lysine residues to alter crystallization properties of proteins. Methods Enzymol. 1997, 276, 171–179. (b) Rayment, I.; Rypniewski, W. R.; Schmidt-Base, K.; Swirth, R.; Tomchick, D. R.; Benning, M.; Winkelmann, D. A.; Wesenberg, G.; Holden, H. M. Three dimensional structure of myosin subfragment-1: A molecular motor. Science 1993, 261, 50. (37) Blundell, T. L.; Johnson, L. N. Protein Crystallography; Academic Press: New York, 1976.
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