Protein Crystallization: Theory and Practice Excerpts from: Structure and Dynamics of E. coli Adenylate Kinase A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY Michael B. Berry W. M. Keck Center for Computational Biology Rice University Houston, TX 9/17/95 Copyright 1995 ------------------------------------------------------------------------ Disclaimer This is my version of a basic-to-advanced guide to the crystallization of proteins. Actually, it's just excerpts from
Transcript
Protein Crystallization:Theory and Practice
Excerpts from:
Structure and Dynamics of E. coli Adenylate Kinase A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE DOCTOR OFPHILOSOPHY
Michael B. BerryW. M. Keck Center for Computational BiologyRice UniversityHouston, TX9/17/95
This is my version of a basic-to-advanced guide to thecrystallization of proteins. Actually, it's just excerpts from
Chapters 2 and 3 of my dissertation compiled together under asingle heading with a few new sections. The methodology coveredherein varies from basic to voodoo black magic (hair of dog), butis by no means complete. This guide is intended mostly to providebackground in crystallization theory and to help in thecrystallization of unknown or otherwise resistant proteins.Hopefully this will be helpful. Please pay attention to the warningsin the text when dealing with some of the more toxic substancesused in the crystallization of proteins, especially cadmium,thiocyanide, and azide. Use these at you own risk!
Crystallization TheoryCrystallization is one of several means (including nonspecificaggregation/precipitation) by which a metastable supersaturatedsolution can reach a stable lower energy state by reduction ofsolute concentration (Weber, 1991). The general processes bywhich substances crystallize are similar for molecules of bothmicroscopic (salts and small organics) and macroscopic (proteins,DNA, RNA) dimensions. There are three stages of crystallizationcommon to all systems: nucleation, growth, and cessation ofgrowth.
Nucleation is the process by which molecules or noncrystallineaggregates (dimers, trimers, etc.) which are free in solution cometogether in such a way as to produce a thermodynamically stableaggregate with a repeating lattice. Crystallization is known tolower the free energy of proteins by ~3-6 kcal/mole relative to thesolution state (Drenth and Haas, 1992). The formation ofcrystalline aggregates from supersaturated solutions does nothowever necessitate the formation of macroscopic crystals.Instead, the aggregate must first exceed a specific size (the criticalsize) defined by the competition of the ratio of the surface area ofthe aggregate to its volume (Feher and Kam, 1985; Boistelle and
Astier, 1988). Once the critical size is exceeded, the aggregatebecomes a supercritical nucleus capable of further growth. If thenucleus decreases in size so that it is smaller than the critical size,spontaneous dissolution will occur. The process of formation ofnonspecific aggregates and noncrystalline precipitation from asupersaturated solution does not involve the competition betweensurface area and volume (n-mers add to the aggregate chain in ahead to tail fashion forming a linear arrangement), and thusgenerally occurs on a much faster time scale than crystallization.
The degree to which nucleation occurs is determined by the degreeof supersaturation of the solutes in the solution. The extent ofsupersaturation is in turn related to the overall solubility of thepotentially crystallizing molecule. Higher solubility allows for agreater number of diffusional collisions. Thus, higher degrees ofsupersaturation produce more stable aggregates (due to higherprobability of collision of diffusing molecules) and thereforeincrease the likelihood of the formation of stable nuclei. In the caseof a finite number of solute molecules, this condition generallyresults in the production of a large number of small crystals. Atlower solute concentrations the formation of individual stablenuclei increases in rarity, thus favoring the formation of singlecrystals.
Crystal growth generally starts at solute concentrations sufficientfor nucleation to occur, and continues at concentrations beneath thenucleation threshold. The rate of growth is determined by acombination of the nature of the growing crystal surface and thediffusional rate. Addition of molecules to a rough surface requiresless energy than addition to a smooth surface, where surfacenucleation is required for addition.
According to Periodic Bond Chain theory, (Boistelle and Astier,1988), three different types of growth faces exist: flat faces,stepped faces, and kinked faces. Flat faces require two dimensional
nucleation (the formation of growing sheets of molecules) in orderto induce growth, and thus grow the slowest. Stepped faces growas columns of molecules, which requires only one dimensionalnucleation, and thus have intermediate growth rates. Stepped facestypically occur as a result of a crystallographic screw axis causingspiral growth patters to occur at the surface of the crystal. Finally,kinked faces are growth sites which do not require nucleation topromote further growth, and therefore grow faster than the othertwo face types. Thus, the type of the growing crystal face (flat,stepped, or kinked) strongly influences the rate at which crystalgrowth occurs.
The growth of crystals from nuclei is also strongly influenced bydiffusional and convection effects. As with nucleation, increasedsolubility results in increased growth rates. Again, this is a functionof the rate at which protein molecules reach the growing surface ofthe protein crystal. Feher and Kam, through the use of ultravioletmicroscopy, have been able to demonstrate the regionssurrounding growing crystals to be lowered in proteinconcentration relative to the surrounding solution (Feher and Kam,1985). The rate of diffusion of proteins in and out of these halosaround the growing crystal provides a growth limiting factor.
The formation of halos due to lowered solute concentration aroundgrowing crystals also has the effect of producing density gradientsin these areas. These in turn (under the effects of gravity) result inthe formation of convection currents which may dominate the rateof simple diffusion and adversely effect crystal growth(Rosenberger, 1986). As the formation of concentration gradientsaround growing crystals is directly proportional to the rate at whichmolecules add to the surface (the crystal growth rate), slowergrowth results in decreased convection currents. This may beaccomplished by growing crystals in porous gel medias (Robertand Lefaucheux, 1988). Crystal growth in zero (effective) gravity
may also be used to remove convective and sedimentary effects(Littke and John, 1986; DeLucas et al., 1986).
Cessation of growth of crystals can occur for a multitude ofreasons. The most obvious is the decrease in concentration of thecrystallizing solute tothe point where the solid and solution phasesreach exchange equilibrium. In this case, the addition of moresolute can result in continued crystal growth. However, somecrystals reach a certain size beyond which growth does not precedeirrespective of solute concentration. This may be a result either ofcumulative lattice strain effects or poisoning of the growth surface.
Lattice strain effects in tetragonal lysozyme crystals have beendemonstrated by Feher and Kam and coworkers (Feher and Kam,1978). Halved crystals of hen egg white lysozyme, when placed infresh crystallization solutions, grew to the exact same size as theoriginal crystal. This suggests that the long range propagation ofstrain in the lattice effectively prevents addition of molecules to thesurface once a certain critical volume is reached. Crystals affectedby lattice strain are therefore inexorably size limited.
Poisoning of growing faces occurs when foreign or damagedmolecules are incorporated into the growing crystal face resultingin successive defects which interrupt the crystal lattice. Anexample of this might be the incorporation of a proteolyticallyknicked protein onto the face of an otherwise perfect proteincrystal. If the knicked molecule is unable to form the same latticecontacts with newly added molecules as would the perfect protein,then its incorporation will cause local defects in the growinglattice. Since the growth of crystal lattices typically selects forperfect over damaged or incorrect molecules, the concentration ofthese defective molecules relative to perfect molecules tends toincrease as growth proceeds. Thus, as crystals grow larger thelikelihood of incorporation of defective molecules into the latticeincreases (also the increase in surface area contributes). Sato and
coworkers have used laser scattering tomography to visualizelattice defects in large crystals of orthogonal hen egg whitelysozyme (Sato et al., 1992). Their results demonstrated theoccurrence of both point and inclusion defects not only at thesurface, but within the bulk of the crystal itself.
To increase the size of surface poisoned crystals, the poisonedsurfaces must be removed by partially melting the crystal. Thisprovides an unpoisoned surface which can be used for furthergrowth in the presence of more solute. A method of applying thistechnique to protein crystals has been designed by Thaller andcoworkers (Thaller et al., 1981).
Solution properties influencing crystallization Crystallization ofmacromolecules is a paradigm on par with protein folding as to thenumber of possible substates which exist in excess of the globalminimum where perfect crystals form (Feher and Kam, 1985). Thereasons behind the high degree variability in the crystallization ofmacromolecules are manifold and include: 1) the high degree ofmobility at the surface and as a whole for large molecules, 2) theelectrostatic nature of macromolecules (i.e. their composition as arandom assortment of mobile point charges embedded in a flexiblelow dielectric medium), 3) the relatively high chemical andphysical instability of macromolecules (unfolding, hydrationrequirements, temperature sensitivity), and 4) molecule specificfactors (such as prosthetic groups and ligands). These factorscontribute greatly to the difficulty and crudity in current attemptsto predict crystallization behavior by de novo calculations even forrelatively small and stable proteins like lysozyme (Durbin andFeher, 1991). The above factors are also reflected in the generaltrend towards greater crystal disorder (and lower resolution of
diffraction data) as molecular size increases due to the increasedflexibility and disorder in these crystals, although this is by nomeans universally applicable (see Abrahams et al., 1994)
By and far the most important factor in crystallization is the purityof the sample to be crystallized. A 1 ppm contaminant in a typical10-20 mg/ml protein solution amounts to ~109 molecules (Carter,1988). Although crystal growth, by nature, tends to excludeimpurities (such as in the case of rabbit muscle aldolase whichcrystallizes out of homogenized muscle preps at 52% ammoniumsulfate; Taylor et al., 1948), the presence of high concentrations ofimpurities in small volumes, as are present in vapor diffusion anddialysis experiments, will undoubtedly lead to contamination of thecrystal lattice, and ultimately poorer crystals. Lin and coworkersrecommend the use of FPLC (or HPLC) for general purification ofproteins and to assure homogeneity on both macroscopic andmicroscopic levels (Lin et al., 1992). Lin notes several cases wherethe use of FPLC techniques has improved the reproducibility ofcrystallization as well as the maximum resolution to which proteincrystals diffract.
As noted earlier, molecules crystallize from metastablesupersaturated solutions as a means of lowering the overallsolution free energy. Chemical precipitants are by and far the mostwidely used method of achieving supersaturation ofmacromolecules in order to induce crystallization. In general, themain influence of these compounds is on the solvent (e.g. bulkwater) rather than on the solute (the protein), with the notableexception of dye precipitants. For crystallization of proteins, themajor classes of precipitants may be divided into six categories:salts, high molecular weight straight chain polymers (e.g. PEG),MPD, organic solvents, sulfonic dyes, and deionized water(McPherson, 1990; Arakawa and Timasheff, 1985). Although thefollowing discussions of the individual traits of these six categoriesuse proteins as examples, most of these precipitants are applicable
to other macromolecules including RNA, DNA, andpolysaccharides.
Salts Salts are by far the most common precipitant type used tocrystallize macromolecules. Historically they have been the mosteffective precipitants tried, although, until recently, there were fewother options (McPherson, 1991). Unfortunately, salts generallyhave the drawback of increasing the mean electron density of thecrystallization solution which decreases the signal to noise ratio forcrystallographic data. Salts also have a tendency to interactstrongly with heavy atom compounds, making crystalderivitization for M.I.R. phasing difficult.
The efficacy of a particular salt as a precipitant is proportional tothe square of the valences of the cations and anions which make upthat salt. Typically, the anion is the more important species thanthe cation, as in general, innocuous monovalent cations are used toavoid forming strong cation/protein complexes which tend to occurwith polyvalent cations (especially transition metal ions). Theability of a salt to precipitate proteins can be generally describedby the Hofmeister series:
Anions and cations which are weakest in the Hofmeister seriestypically have the effect of salting in rather than salting outproteins. In practice the PO43- anion does not exist in solutionwithin the range of pH typically used for crystallization trials, andis present instead as HPO42- and H2PO4-. Also, the NH4+ cation
generally loses H+ above pH 8.0 and boils off as NH3, makingNH4+ salts difficult to use at high pH, as well as making the pHhighly unstable. An incomplete list of effective salt precipitants (indescending order of efficacy), their maximum concentrations, andgenerally effective ranges are shown in Table 1.1. (It should benoted, however, that there is a general trend for larger molecules[up to viruses; see Sehnke et al., 1988] to crystallize at increasinglylower precipitant concentration as the molecular weight of thespecies to be crystallized increases. Thus the values given in thistable are probably applicable for 15-50 kDa Mw proteins.).
Two general categories of salts exist, those which mainly interactwith water (non-chaotropic salts), and those which mainly interactwith the protein (chaotropic salts). Non-chaotropic salts arepreferentially hydrated with respect to protein solutes. The effectof this is to increase in surface tension of the solvent surroundingthe macromolecule, thus dehydrating the protein's surface andcreating excluded volume effects, which force solute molecules toform close interactions with each other. Solutions of these saltscharge shield proteins from one another by increasing the solventdielectric, which in turn further facilitates close protein-proteininteractions. Binding of non-chaotropic salts by the protein doesnot generally play a role in crystallization, although in some casessalt ions (particularly sulfates and phosphates) are seen bound bymacromolecules in crystal structures, and occasionally act aslattice contacts between molecules in the crystal. Typically, thesesalts increase the stability of macromolecules in solution (Scopes,1994).
Chaotropic salts are generally not used for crystallization due totheir tendency to salt in macromolecules and to induce unfoldingthrough interactions with secondary structural elements of proteins.However, Ries-Krautt and Ducruix have discovered that, at
reasonable concentrations (~200 mM) and low pH (~4.0-5.0),potassium thiocyanate (a chaotropic salt) acts as precipitants byinteracting with and neutralizing positively charged residues on thesurface of proteins (lysine, arginine, histidine, and the aminoterminus; Riès-Kautt and Ducruix, 1991). It was further noted thatthe addition of small amounts of KSCN (~10 mM) to non-chaotropic salt precipitants (phosphate) increased the rate ofcrystallization substantially (WARNING: SCN- ENDUCESPSYCHOSIS, don't eat it!). Other chaotropic salts (such as KI,urea, and guanididium-HCl) may be equally effective asprecipitants or additives at concentration low enough to preventdenaturation.
Polymers The use of high molecular weight linear polymers asprecipitating agents was pioneered by Polson and coworkers whotried a variety of polymers including polyethylene glycol, dextran,polyvinyl alcohol, and polyvinyl pyrrolidone (Polson et al., 1964).Of these, polyethylene glycol (PEG) was found to be the mosteffective both by way of precipitating ability and costeffectiveness. PEG's are produced in a variety of molecularweights, ranging from 200 (~3 monomers) to in excess of 1 million(~15000 monomers), and as mono- and di- methyl ethers. Likesalts, PEG's compete with protein solutes for water and exertexcluded volume effects (which vary according to the length of thepolymer). However, unlike salts, PEG's decrease the effectivedielectric of the solution, which increases the effective distanceover which protein electrostatic effects occur. Solutions ofpolyethylene glycols have mean electron densities roughlyequivalent to water and do not generally interact in a deleteriousmanner with heavy atom compounds, thus making themparticularly well suited for macromolecular crystallization. PEG'swith molecular weights less than 1000 are typically liquids and aregenerally used at concentrations above 40% v/v. PEG's withmolecular weights above 1000 are generally solids and are used in
the 5-50% w/v concentration range. All PEG solutions should bemade with the inclusion of ~0.l% Na azide (which, incidentally, isalso highly toxic to humans) to prevent bacterial growth. Also,buffering of high concentration (40%) PEG solution with Nacitrate at concentrations above 100 mM tends to cause theformation of phase transitions and color changes in the PEGsolution, which suggests some form of reaction (probably crosslinking by the citrate), and thus should be avoided.
MPD MPD is a small polyalcohol (2-methyl-2,4 pentane diol)which has properties midway between those of low molecularweight PEG's and organic solvents. MPD functions as a precipitantby a combination of activities, including competition for water,hydrophobic exclusion of protein solutes, lowering the solutiondielectric, and detergent-like effects. It is generally used inconcentrations in excess or 40% v/v with water/buffer, and tends tocause phase transitions in the form of coacervate droplets whichare enriched in protein concentration (synonymous with those inRay and Bracker, 1986). As with PEG, the use of MPD as aprecipitant produces a low electron density solution which does notgenerally interact with heavy atom compounds.
Organic solvents Historically, organic solvents have typically beenused as precipitants for protein crystallization as much by chanceas by design (McPherson, 1988). Crystallization by exposure toorganic solvents is occasionally seen during protein purification,typically in the presence of common solvents such as ethanol,methanol, acetone, isopropanol, DMSO, or tert-butanol(McPherson, 1990). Due to their hydrophobic nature, organicsolvents cause phase transitions similar to those formed in thepresence of MPD and lower the bulk dielectric of the solvent.Organic solvents also tend to cause protein denaturation unlessthey are used at temperatures at or below 0 degrees C.
Sulfonic dyes Sulfonic dyes are typically large planar polycycleswith attached sulfonate groups. Initial studies by Lovrien andcoworkers suggest that these molecules may specifically interactwith the surfaces of proteins by way of the dyes sulfonate groups(Conroy and Lovrien, 1992; Lovrien et al., 1993). The planarpolycycle of protein/dye complex greatly alters the solubility of theprotein triggering precipitation and occasionally crystallization ofthe protein/dye complex. Lovrien suggests that a ratio of ~1-5 dyemolecules per protein molecule is sufficient forprecipitation/crystallization. NOTE, many sulfonic dyes areextreme carcinogens and/or teratogens due to their tendency tointercalate intoDNA.
Deionized water Dialysis of protein solutions against deionizedwater as a method of crystallization takes advantage of therequirement of proteins to be surrounded by a cloud of positivelyand negatively ions in order to be soluble (the Donnan effect). Ifthis cloud of anions and cations is removed by dialysis, the proteinwill attempt to surround itself with whatever charged species arepresent, which, in this case, will be other protein molecules. Theformation of three dimensional lattice structures (e.g.crystallization) is energetically favored as these arrangementsgenerally entail a more complete ionic atmosphere around eachmolecule in the lattice.
Although all of the chemical precipitants discussed above haveworked for one system or another, oftentimes their individualeffects are too severe for crystallization to occur and instead theprotein solute leaves the supersaturated state by way ofprecipitation rather than crystallization or may form poor one ortwo dimensional crystals. In such cases, combinations of
precipitants (i.e. salts and PEG's, MPD and salts, salts andorganics, etc.) may produce larger, better diffracting crystals, andoccasionally new space groups. Some of the best combinations(based on results of crystallization trials) are outlined in Table 1.2.
A trend which can be seen in all of these solutions is thecombination of either a high dielectric species (major precipitant)with a species which lowers the dielectric (minor precipitant, saltswith PEG/MPD/organics), or a low dielectric species (majorprecipitant) with species which raise the dielectric (minorprecipitant, PEG/MPD/organics with salts). Both types ofcombinations may effectively maximize size exclusion and watercompetition effects while preventing intermolecular chargerepulsion by increasing the solvent dielectric, thus improving boththe likelihood of crystallization and the quality of the crystalswhich do appear. Additives may also act to salt in or salt outmacromolecules which are insoluble or especially soluble in themajor precipitant used. Biphasic solutions (e.g., such as thosecreated by mixing high concentrations of salts with highconcentrations of high molecular weight PEG's) may also beeffective in triggering nucleation and crystallization (Kuciel et al.,1992; Ray and Bracker, 1986; Garavito et al., 1986)
Addition of metal ions (in particular, cadmium (Cd2+), cobalt(Co2+), and manganese (Mn2+)) to crystallization solutions can beused as a form of "intermolecular glue" to either trigger nucleationor improve crystallization (McPherson, 1991). Cadmium ions tendto form strongly covalent complexes between two adjacentcarboxylic acid groups in a square planar type arrangement with allfour carboxylic acid oxygens being involved in bonding. This typeof bond can act as a lattice contact in a crystal or as a nucleatingfactor for protein crystal growth. Manganese has similar (thoughweaker) interactions, while cobalt ions preferentially interact withamines (lysines and histidines). Typically, 1 to 5 mMconcentrations of CdSO4, 1 to 50 mM MnSO4, or 1 to 50 mMCoSO4 are used either alone or in conjunction with anotherprecipitant. Caution should be exercised when working withtransition metal ions due to their general toxicity. Cadmium isespecially dangerous as, with beryllium and thallium, it is one ofthe three most toxic elements known.
Two other chemical factors which can be used to initiate orenhance crystallization are viscosity altering compounds and anti-twinning/solubilizing compounds (detergents and ethers). As notedin the section on crystallization theory, the rate at which crystalsgrow often affects the overall quality of the crystals due to theinclusion of defective or misaligned molecules. By inclusion ofviscosity altering compounds (notably glycerin) in thecrystallization mother liquor the rate of crystal growth may beretarded. In attempts to crystallize the 2 ADP complex of E. coliadenylate kinase this did result in larger crystals, although theywere as poorly ordered as smaller crystals of the samemorphology. Generally, 1% glycerin is sufficient to greatly slowthe rate of crystallization and/or nucleation.
The use of detergents as additives in crystallization mother liquorshas been utilized mostly in attempts to crystallize membraneproteins (Garavito et al., 1986; Gros et al., 1988). The hydrophobictail of the detergent molecule binds to the hydrophobic areas of theprotein which are usually embedded in the membrane, thussolubilizing these areas through the exposed hydrophilic headgroups of the detergent molecules. At lower concentrations,detergents, as well as simple ethers like dioxane, can be used inconjunction with other precipitants either to increase the solubilityof poorly soluble molecules or to improve crystallization habit, andparticularly to reduce or eliminate twinning (Bergfors, 1993;McPherson et al., 1986). In one case, McPherson and coworkersreported an entirely new crystal form of a protein due simply to theaddition of 0.1-1% BOG (beta-octyl glucoside, a mild detergent) tothe crystallization conditions. Crystal size also appeared to beincreased, while nucleation decreased. The use of detergents is notentirely harmless in all cases, as at too high a concentration theyquickly denature myoglobin, probably either by solubilizing theoily heme pocket or by solubilizing the heme itself.
The most important factor in crystallizing proteins other than thechemical composition of the mother liquor is its pH. For someproteins (e.g. the closed conformation of E. coli adenylate kinase),crystallization occurs over a very broad range of pH with little byway of variation in crystal morphology. However, it is far moretypical for crystallization to occur over a fairly narrow range (< 1pH unit). Crystal morphology, including various twinned andpolynucleated growth forms, often is directly related to pH.Typically, there is a gradual improvement in crystal morphology asthe proper pH is approached, and a fall off of crystal quality oneither side of the optimal condition. In some cases, the pI of theprotein is the pH at which the best crystals grow, so this should, ifpossible, be known in advance and tried as a condition.
Temperature is another factor which is of considerable import incrystallization of protein. Crystallization of macromolecules hasbeen accomplished in a range of roughly 60 degrees C to < 0degrees C, although the vast majority of molecules are crystallizedeither at 4 degrees C or 22 degrees C (room temperature). Lowtemperature tends to act as a preservative for sensitive proteins aswell as an inhibitor of bacterial growth. Solubility of proteins insalt solutions tends to increase at low temperatures (4 degrees C),while in PEG and MPD solutions, protein solubility generallydecreases with decreasing temperature. By increasing ordecreasing either precipitant or protein concentration,crystallization should, at least in theory, be possible at either roomtemperature or 4 degrees C, although the kinetics of crystallizationcan be expected to vary in accord with temperature. Heating,melting, and cooling of crystals or aggregates may also be tried inorder to enlarge crystals, although this is generally unsuccessful.Overall, the mantra according to McPherson and others is to tryboth 4 degrees C and 22 degrees C, protein permitting(McPherson, 1992; Carter and Carter, 1979; Bergfors, 1993).
Chemical/biochemical modification of proteins is another factorwhich may be used to change crystallization conditions.Electrostatic surface characteristics play a large role in dictatingwhether a protein crystallizes or not. Thus, modification of surfacecharges by either chemical (derivitization) or biochemical(mutagenesis) means can provide crystals where none were knownbefore (Rayment et al., 1993) or provide crystals in new spacegroups, possibly with better resolution (McElroy et al., 1992).Rayment reductively methylated lysines of chicken muscle myosinsubfragment-1 to improve crystallization and alter the space groupin which the protein crystallized.
Biochemical modification in the form of site directed mutagenesisof surface residues has been utilized by McElroy and coworkers toimprove the crystallization characteristics of human thymidylatesynthase (McElroy et al., 1992). The protein initially crystallized insuch a way as to make it impossible to interpret the active site inelectron density maps due to disorder. Therefore, McElroy andcoworkers created single point mutations at non-conserved surfaceresidues at twelve different sites (one per mutant protein) whichwere designed either to neutralize charges (mutation toasparagine), reverse charges (arginine and lysine to glutamate oraspartate, and vice-versa), or add charges (cysteine, proline,leucine, and glutamine to aspartate, glutamate, or lysine). Thecrystallization behavior for each point mutation was completelyunpredictable and multiple new crystal forms resulted. Whereasthe original crystal form was trigonal (P3121), single point mutantscrystallized in monoclinic (C2), orthorhombic (C222), andtetragonal (P41212) space groups. The tetragonal space group wasconfirmed as having a well defined binding pocket. What isinteresting in these results is that an example of each type ofmutation (neutralization, reversal, or addition of charge) producedan alternate space group to the original trigonal form. As thistechnique is far more controllable (although considerably moreexpensive) than chemical modification, it suggests a possible
avenue for producing or improving crystals for proteins for whichrecombinant expression systems exist.
Physical methods of crystallizationFour methods are commonly employed to affect supersaturation inthe crystallization of macromolecules: vapor diffusion, freeinterface diffusion, batch, and dialysis. Although each of thesetechniques achieves supersaturation of the particularmacromolecule to be crystallized, the means by whichsupersaturation is achieved in each case varies greatly. Adiscussion of each of these techniques is presented below.
Vapor diffusion The vapor diffusion technique utilizes evaporationand diffusion of water between solutions of different concentrationas a means of approaching and achieving supersaturation ofmacromolecules. Typically, the solution containing themacromolecule is mixed 1:1 with a solution containing theprecipitant at the final concentration which is to be achieved aftervapor equilibration. The drop containing the 1:1 mixture of proteinand precipitant (both of which have been diluted to 1/2 the originalconcentration by mixing with the other) is then suspended andsealed over the well solution, which contains the precipitant at thetarget concentration, as either a hanging or sitting drop. [Glasscapillaries containing protein and precipitant at concentrationsbelow that required for crystallization can also be vaporequilibrated against a well solution in a sealed test tube. DeMatteiand coworkers have shown capillary based vapor equilibration tooccur at rates up 102 times slower than drop based methods,resulting in improved crystals (DeMattei et al., 1992).] Thedifference in precipitant concentration between the drop and thewell solution is the driving force which causes water to evaporatefrom the drop until the concentration of the precipitant in the drop
equals that of the well solution. Since the volume of the wellsolution is much greater than that of the drop (1-3 ml as compared1-20 ul) its dilution by the water vapor leaving the drop isnegligible.
Fowlis and coworkers have demonstrated that the rate of vaporequilibration in normal (1 g) gravity is dependent strictly on therate of vapor diffusion of water in the space separating the dropand the well (Fowlis et al., 1988). Due to convection effects(caused by the increased concentration of the precipitant at theedge of the drop as water evaporates), the rate of diffusion of awater molecule in the suspended drop (in solution) is actuallygreater than that of the vaporized water molecule. In microgravityexperiments (zero effective g), the rate of equilibration is basedsolely on the rate of diffusion of water in the drop until crystalnucleation, at which point in time Marangoni convection (due toconcentration gradients or surface tension around the crystal)increases the rate of equilibration (Provost and Robert, 1991).Provost and Robert report that the use of simple agarose gels canoffset convection currents under normal gravity producingimproved results with hanging drops.
Vapor diffusion is the optimal technique to use either whenscreening a large number of conditions (by varying thecomposition of each well solution) or when dearth of proteinprevents the use of other methods. Furthermore, this method can beused to increase or decrease the concentration of protein in theequilibrated state relative to its initial concentration. This is doneby varying the volume of protein mixed with the well solutionwhen the drop is initially setup. Since the drop equilibrates so thatthe precipitant concentration matches that of the well solution, thefinal volume of the drop will always equal that of the initialamount of well solution mixed with the protein. Thus, if theprotein solution is mixed in a 2:1 ratio with the well solution, the
concentration of the protein at vapor equilibrium will be doubledrelative to its initial value.
One of the drawbacks to vapor equilibration is that it tends to formsmaller crystals than other methods. This may be due to small dropvolumes limiting the quantity of crystallizable solute present orcreating a higher level of impurities relative to other techniqueswhich utilize larger volumes. As discussed earlier in section 2.2, ascrystals grow, the concentration of defective molecules increasesrelative to perfect molecules (which are being selected for by thecrystal). When this factor is combined with the higher probabilityof impurities diffusing to the face of the crystal (due to the smallervolumes), the likelihood of inclusion of defects into the growingcrystal increases. Thus, the production of X-ray quality crystalsmay be better suited to the use of batch, free interface diffusion, ordialysis techniques which utilize larger solution volumes atequilibrium.
Free interface diffusion Layering of a low density solution ontoone of higher density, usually in the form of concentrated proteinonto concentrated salt, can be used as a means of growing largecrystals. Nucleation and crystal growth generally occurs at theinterface between the two layers, at which both the concentrationof salt and the concentration of protein are at their highest values.The two solutions slowly intermix over time, and should be madeup so that at equilibrium, (at which point in time both solutions arediluted to some fraction of their initial values), the concentration ofthe precipitant is still high enough to promote crystal growth. Sincethe solute to be crystallized must be concentrated, this methodtends to consume fairly large amounts of protein. This method wasused by Steve Althoff to grow large crystals of the E. coliadenylate kinase/Ap5A complex (Althoff, 1987; Althoff et al.,1988).
Batch In the batch method, concentrated protein is mixed withconcentrated precipitant to produce a final concentration which issupersaturated in terms of the solute macromolecule and thereforeleads to crystallization. This can be done with up to ml amounts ofsolution and typically results in larger crystals due to the largervolumes of solute present and the lower chance of impuritiesdiffusing to the face of the crystal. Needless to say, this techniqueis by far the most expensive in terms of consumption of the solutemacromolecule, and thus should not generally be used to screeninitial conditions for crystallization.
Dialysis Dialysis techniques utilize diffusion and equilibration ofsmall precipitant molecules through a semipermeable membrane asa means of slowly approaching the concentration at which themacromolecule solute crystallizes. Initially, the solute is containedwithin the dialysis membrane which is than equilibrated against aprecipitant solution. Equilibration against the precipitant in thesurrounding solvent slowly achieves supersaturation for the solutewithin the dialysis membrane, eventually resulting incrystallization. Dialysis tubing can be used by itself, in the case oflarge amounts of protein being available, or used to cover theopening of a dialysis button, allowing diffusion of the surroundingsolvent in to the solute through the dialysis membrane. Dialysisbuttons themselves come in a variety of sizes from 7-200 ul. Theadvantage of dialysis over other methods is in the ease with whichthe precipitating solution can be varied, simply by moving theentire dialysis button or sack from one condition to another.Protein can thus be continuously recycled until the correctconditions for crystallization are found (Carter et al., 1988). Onedrawback of this method is that it does not work at all withconcentrated PEG solutions, as they tend to draw all the water outof the button or sack faster than PEG dialyzes across themembrane, thus resulting in precipitated protein.
Strategies for screening crystallization conditions Crystallization of uncharacterized or even well characterizedmacromolecules is not nearly as straightforward as it might seem.There are a vast number of possible conditions which must beanalyzed for their ability to produce crystals.
To exemplify this, consider an initial screen where only fivedifferent precipitants are to be used. (This is a conservative numberto start with. Through combination of the major categories withadditives, there are probably hundreds of viable precipitants, eachwith their own unique ability to influence or triggercrystallization.) Each of these five precipitants are set up (with themacromolecule) at four concentrations, and each of these groups isset up at a different pH, from 4-9 at one pH unit intervals. At thispoint 120 conditions are to be tried. If two different temperatures(say, 4 degrees and 20 degrees C) and two macromoleculeconcentrations are tried for all these conditions, the number ofinitial trials approaches 500. Needless to say, the more variableswhich are tested, the greater the expense in terms of labor andmacromolecule, all of which is for naught if the conditions areincorrect for crystallization to occur.
To add to this, the kinetics of nucleation and crystal growth vary ina seemingly random fashion. Thus, a condition which is viable forcrystal growth but unstable for nucleation may take days to yearsto produce crystals, whereas another condition might producecrystals in a matter of minutes. Worse yet, microscopiccrystallization might occur but go unnoticed due to its similarity inappearance to precipitated solute. (The appearance of crystals ininitial screens is only rarely obvious. Most of the timecrystallization is completely inobvious.)
Various strategies have been set forth to initially screen conditionsfor crystallization. These vary from somewhat rational approaches(screening at the pI) to highly regimented approaches (successivegrid screening) to analytical approaches (incomplete factorials,solubility assays, perturbation) to randomized approaches (sparsematrices). Each of these strategies has their own particular usebased on whether the protein is unknown, well known, orsomewhat known (mutated). A short explanation of the variousapproaches with references is provided below.
Screening at the pI The pI of a macromolecule (the pH at which ithas no net charge) is its point of lowest solubility. Therefore, it isrational to assume this to be a point where crystallization mayoccur. Screening at the pI may be done either by dialysis againstlow concentrations of buffer (<20 mM) at the appropriate pH (seesection 2.3.2 on deionized water), or by the use of conventionalprecipitants. In the case of adenylate kinases, however, each of thefour liganded states has a different pI, and usually only the pI ofthe unliganded state is known, requiring the computationaldetermination of the pI for the unknown states.
Grid screening Grid screens are typically based on twodimensional matrices with the two different factors to be tested asthe two axes. Screening takes place as an iterative process, startingwith a course grid over a wide range and ending with a very finegrid over a narrow range. This type of procedure lends itself wellto robotic automation (Cox and Weber, 1988). Typically, the firstscreen conducted in this method is precipitant concentration vs.pH, as these are generally the two most important factors in thecrystallization of macromolecules. The result of the initial screen isthen graded on the lack or presence, as well as the type, ofprecipitant, which can be flocculent (fluffy clouds), granular(amorphousballs), dendritic (string), or crystalline (from micro
needles and needle balls to perfect crystals). A range of conditionsis then prepared which includes and brackets the best initialcondition, and this procedure is repeated until the two parametergrid converges on a best condition. This condition then can be usedas an initial starting place to test additional factors (temperature,additives). This method works well if crystals appear early in thescreening process, but otherwise becomes expensive in terms ofprotein due to the need to exhaustively sample a wide range ofconditions.
Incomplete factorials Carter and Carter pioneered this approach asa "rational exploration of cause and effect relationships governingthe crystallization of proteins" (Carter and Carter, 1979). Theincomplete factorial approach is to take an initial set of ~20conditions, randomly assign combinations of these factors asindividual experiments, grade the success of the results of eachexperiment based on an arbitrary objective scale, and statisticallyevaluate the effects of each of the twenty factors on thecrystallization trials (Carter et al., 1988). Thus, the effects offactors such as pH, temperature, precipitating agent, and cationscan be determined precisely with minimal subjective interpretation.These experiments are carried out in dialysis buttons so thatprotein can easily be recycled (by dialysis against buffer to removeprecipitant), and so that each experiment can be driven tocompletion by changing the precipitant concentration until eitherprecipitation or crystallization occur. In the two sets of trialsreported by Carter and coworkers, final conditions yielding highquality crystals, sometimes in multiple space groups, weredetermined within 35 trials.
A similar approach is espoused by Stura and coworkers whichutilizes a solubility "footprint" to rationally determine the directionof further trials (Stura et al., 1991). An initial series of sixprecipitants at four different concentrations and three pH's yields
an initial direction for further incomplete factorial searches basedon the degree of solubility in each condition. This method can beused to determine conditions at which the macromoleculecrystallizes even if the initial footprint does not yield crystallinematerial.
Perturbation One or two dimensional crystals which cannot beimproved upon by further grid iteration have appeared severaltimes in the course of my attempts to crystallize mutant spermwhale myoglobins and recombinant human hemoglobins (Whitakeret al., 1995). In these cases,------------------------------------------------------------------------
Table 1.3
Additives for perturbation screening(In addition to the major precipitant, e.g. ammonium sulfate)
perturbation assays have been particularly effective in producingusable three dimensional crystals. The perturbation approachcombines the condition which yields the one or two dimensionalcrystals combined individually with a series of additives designedto test the effects of altering the structure of bulk solvent and alsothe solvent dielectric. Categorical list of additives are shown inTable 1.3.
Sparse matrices Jancarik and Kim first described the use of asparse set of conditions for initial crystallization trials based onconditions which were known to have crystallized proteins [theCrystal Screen sold by Hampton is made up of solutions suggestedby Jancarik and Kim] (Jancarik and Kim, 1991; see also:McPherson, 1992). The idea behind sparse matrix trials is toprovide a broad enough sampling of parameter space by random(or near random) combination of conditions to yield initial crystalswhich then may be improved upon. This is also a very cheapmethod (if it works) in terms of consumption of the
macromolecule, and therefore is preferable under conditions wherethe quantity of macromolecule is the limiting factor.
In attempts to crystallize recombinant human hemoglobins, I usedthe program sparse which I wrote (sparse.f, in FORTRAN 77)which reads in a series of pH's, precipitants, and additives andoutputs a series of randomized combinations. Several of theconditions created by the program resulted in improved crystals.The program utilizes an initial numerical seed based on the dateand time for random number generation, and therefore each timethe program is run it outputs a completely new series of conditions.
It is to be stressed that the best way to maximize the rate of initialsparse matrix screening is to keep an inventory (e.g. solutionnumber, main precipitant, buffer, pH, additive, concentrations) ofall solutions which have been made and use these in initial trials.Solutions should thus be made with 0.1% Na azide (WARNING:TOXIC, don't eat it!) to prevent bacterial growth. It should benoted that Na azide will bind to the iron of hemin.
A cheap, fast, and effective initial screen The above screen is anintegration of the footprint screen of Stura with a grid approach,plus some sparse conditions thrown in as well. It has been effective
numerous times in crystallizing unknowns. It requires two Linbrotrays and the use of hanging or sitting drops, although tray 2 can beset up weeks after tray 1, to conserve protein. Wells 31-48 of tray 2should be chosen randomly from a list of solutions or generatedusing sparse methods, and should cover the range of precipitants,additives, and pH's (emphasis 5.0-9.0).
SeedingSeeding is a method of insuring or triggering (in the case ofheterogeneous nucleation) nucleation and crystal growth (Sturaand Wilson, 1990, Thaller et al., 1981; McPherson, 1988).Commonly a protein crystal or crystalline aggregate is placed in astorage solution and then crushed to a fine powder (producing alarge number of microscopic crystals) and mixed to make a seedsolution. The seed solution is then added to the supersaturatedcrystallization trial (e.g. added to the equilibrated hanging drop orbatch tube) thus ensuring the presence of nuclei for crystal growth.If no crystalline material is available for a particular protein, seedsolutions from related proteins (e.g. the same protein, but from adifferent species) may sometimes trigger crystallization of thetarget protein.
Microanalytical seeding This technique is designed to analyze theresults of crystallization trials in which some or all of theconditions failed to yield crystals. It is best done with hanging orsitting drops but may be done with other methods as well. In short,high concentrations of seeds are added to all trials and the resultsare judged based on the morphology of the resultant crystals. In
general, nucleation will occur at the site of seed introduction andwill occur from a large number of nuclei, resulting in a multitudeof crystals. Seeds may be introduced either by adding 1-2 ul of theseed solution directly to the equilibrated protein solution (and thusdiluting it) or by dipping a hair (from my dog) in the seed solutionand then streaking this across the surface of the drop (streakseeding; see Stura and Wilson, 1990). I prefer the latter methodbecause it is faster and does not dilute the supersaturated solutionto any degree.
Microproduction seeding This is identical in all regards tomicroanalytical seeding except that the seed solution is diluted(with the crystal storage solution, not with water) to the pointwhere the addition of 1-2 ul or streak seeding results in theaddition of only a few nuclei, allowing for the growth of largesingle crystals.
Macroseeding This is a technique whereby small crystals may beenlarged to a size amenable for data collection (Thaller et al.,1981). In short, small crystals are removed from the solution inwhich they were grown, partially melted in a lower concentrationprecipitant wash solution, and then introduced into equilibratedprotein/precipitant solutions in order to continue growth. There aretwo tricks to this process. The first is to insure that the crystal ismelted enough in the wash solution to remove any potential nucleiat its surface, without completely dissolving the crystal. Thesecond is to chose a precipitant and protein concentration whichallows for continued protein growth but not nucleation. The entireprocess can be repeated multiple time to continue to increase thesize of the crystal.
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