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Protein induced morphological transitions in KCl crystal growth Bálint Szabo´ and Tama´s Vicsek Department of Biological Physics, Eötvös University, Budapest, Pázmány Péter sétány 1/A, 1117 Hungary (Dated: February 2, 2008) WeinvestigatedtheformationofKClcrystalsonglasssurfacebyphasecontrast,fluorescent,and atomicforcemicroscopyonthemicrometerscale,andobservedinterestingmorphologicaltransitions 3 as a function of the experimental conditions. The presence of proteins in the solution from which 0 thesaltcrystalsgrowduringthedryingupleadstocomplexmicroscopicpatternsofcrystalssomeof 0 which are analogous tothose commonly observed on themacroscopic scale. Wetested theeffect of 2 tubulin,FITC-labeledalbuminandIgGonthemorphologyofcrystalsgrowneitherslowlyorfast. A rich variety of protein specific and concentration dependentmorphologies was found and described n by a morphological diagram. We give a phenomenological interpretation, which can explain the a J growth of complex patterns. Fluorescent images prove that a protein layer covers the surface of the KCl structures. We propose that this layer reduces the anisotropy of the effective surface 1 tension during growth. The tip splitting fractal regime is attributed to the decrease of anisotropy. 3 Otherpossiblemechanisms,whichcancausemorphologicaltransition,arealsodiscussed. Wefound ] elongated saw-toothed crystals induced by proteins, especially IgG and identified their structure. h p PACSnumbers: 89.75.Kd,47.20.Dr,68.37.-d - o i b I. INTRODUCTION II. MATERIALS AND METHODS . s c 5 µl drops of 5 mg/ml KCl solution containing either i s tubulin (prepared from bovine brain [14]) or FITC-BSA y (bovine serum albumin conjugated with FITC, SIGMA) h orhumanIgG-FITC(immunoglobulinGconjugatedwith p The effect of proteins, especially albumin and IgG on FITC, SIGMA) or no protein were placed on clean glass [ crystal growth has biological relevance. In crystal in- coverslips. Wepreparedsampleseitherbyslowdryingat 3 duced arthritis e.g., gout proteins bind the crystals [1] ∼40 ◦C in air or by fast drying in a strong airflow. Slow v and have significant impact on crystal growth [2]. The drying took ∼5 minutes, airflow dried the surface on the 5 immune response to the appearance of crystals is driven scaleofseconds. Brightfield,phasecontrastandfluores- 0 by the specific IgG-crystal interaction. This interac- cent images were acquired by an inverse Leica DM IRB 0 tion stimulates crystal formation [3, 4, 5]. Although the 9 microscope,40xobjectiveandaNikonCoolprix700digi- rich morphology of several inorganic macroscopic crys- 0 talcameramountedonthemicroscopebyhomemadeop- 2 tals grown on the surface of gels (i.e., in the presence of ticalcoupling. Atomicforcemicroscopy(AFM)imagesof 0 protein) has been studied and discussed [6, 7] up to our tubulincoatedcrystalswerecapturedwithacommercial / knowledge this is the first study of patternformationin- s AFM(TopoMetrixExplorer,SantaClara,CA)incontact c duced by crystal-protein interactions. Moving unstable mode with a soft silicon nitride cantilever (Thermomi- i interfacestypicallyleadtotheformationofcomplexpat- s croscopes, coated sharp microlevers, model No. MSCT- y terns. Surfacetensionoftheboundarybetweenthegrow- AUHW, with typical force constant 0.03 N/m, 20 nm h ingandsurroundingphasesisessentialinpatternforma- nominal radius of curvature) under ambient conditions. p tion [8, 9, 10, 11]. A growth mode of crystals covered : v withathinlayerhasbeenobservedandexplainedbythe i surface tension between the different phases in a metal X III. RESULTS system [12]. The growth of nonequilibrium interfaces r covered by a thin surfactant film was both experimen- a A. Tubulin tallyandtheoreticallystudied[13]. Theanisotropyofthe surface tension has crucial role. Viscous fingering, crystallization, electrochemical deposition, and some other Thefastdryingof0.5mg/mltubulin containingdrops phenomenacanberelativelywelldescribedbyLaplacian of 5 mg/ml KCl solution on glass surface with air- growthwithappropriateboundaryconditions. We argue flow results in a heterogeneous population of patterns that the growth of the numerous different microscopic that we investigated by AFM and phase contrast mi- patterns we observed in our KCl crystallization experi- croscopy. ThemorphologyofKClcrystalscanbesimilar mentsinthepresenceofproteinscanbeexplainedbythe to diffusion-limited aggregatesknown to have fractal ge- change of the boundary conditions of the moving inter- ometry. Dendritic patternswithstableandunstabletips face: theanisotropyofits surfacetensionisdecreasedby aretypical. FIG.1showsapattern,whichbegantogrow proteins. withstabletips,4-foldsymmetry. Afterawhiletipsplit- 2 ting occurs at all the 4 tips. Anisotropy drops dramati- coverslip. Wefoundroundedformsinsteadofthenormal cally at this point. The slow drying of drops containing rectangular shapes of KCl. (Image not shown.) 0.05mg/mltubulin giveselongatedsaw-toothedcrystals similar to the ones induced by IgG discussed below. In highconcentrationtubulininhibitscrystalgrowth,anap- E. Gold substrate parently homogeneous stain can be observed after the drop dries up. We also studied the effect of the substrate surface, whichwasglassinthe aboveexperiments. However,IgG gave similar results on gold substrate to those on glass B. FITC-BSA in case of the slow drying method, albumin induced the appearance of amorphous protein aggregates and blocks We triedto shedlightonthe roleofproteinin pattern of prism-shaped crystals on gold instead of the patterns formation of salt crystals with the use of FITC labeled describedabove. Thisfactindicatestheproteinandsub- proteins. In case of the fast drying procedure 5 mg/ml strate specificity of the phenomenon. FITC-BSA had similar effect to that of tubulin. Various branchingmorphologieswerefound. Tipsplittingcaused fractal growth (FIG. 2). The comparison of bright field IV. DISCUSSION andphasecontrastimageswiththefluorescentonesshow that crystals are covered by a layer of albumin. Protein Proteins have significant impact on KCl crystalliza- concentration dependence of the morphology was stud- tion. Thepresenceofproteinsinthesolutionfromwhich ied withthe slow dryingtechnique. Under a criticalpro- KCl crystals grow during the drying up leads to the for- tein concentration(∼1µg/ml) only blocks of rectangular mation of protein specific and concentration dependent prism-shaped crystals grew corresponding to the protein complex patterns, which we described by a morphologi- free case. In the concentration range of 1-1000 µg/ml cal diagram (FIG. 7). We can give a partial explanation cubic crystal centered structures formed (FIG. 3). This of the diagram. Meanwhile, the solution dries up the observationreinforcethatduringasinglegrowthprocess protein precipitates due to the increasing ionic strength. the symmetry (anisotropy) of the pattern can dramat- This process is known as salting out. A thick layer of ically change due to the change of the local conditions. proteinaggregate(precipitate)coversthesurfaceofcrys- Asthegrowthprocesselongates,theinitial4-foldsymme- tals. Thiscovermeans2interfaces: onebetweentheKCl try with high anisotropy disappears and rather isotropic crystalandtheproteinlayerandonebetweentheprotein growth takes place. In the higher protein concentration layerandtheliquid. Whilethesurfacetensionofthefor- range dendritic crystals grew with stable tips (FIG. 4). mer one γc−p is anisotropic, that of the latter one γp−l is considered to be isotropic. The total surface tension γ of the interface between the growingand surrounding t C. IgG-FITC phases is given by the sum of these two terms: To study the protein specificity of the phenomena we examined the impact of human IgG-FITC on the KCl γt =γc−p+γp−l (1) crystal growth. After the fast drying procedure using 2 mg/ml IgG the typical pattern we found was the elon- The value of γp−l i.e., the isotropic term is significant sinceprecipitationmeansinsolubilityoftheprotein. The gated saw-toothed crystal (FIG. 5). Branching struc- roundedequilibriumshape ofcrystalswith proteincover tures can be observed. The angle between the branches is ∼70◦. Concentration dependence of the morphology reinforces the loweranisotropy of surface tension. In the was investigatedby the slow drying method. Under ∼20 absenceofproteintheextraisotropictermcorresponding to the protein-liquid interface is missing. If µg/ml only blocks of the prism-shaped crystals grew. Abovethisconcentrationupto∼1mg/mlcubicandsaw- γc−p <γp−l, (2) toothed crystal centered structures were observed with an isotropic surrounding pattern. FIG. 6 shows the cen- then an asymmetry arises between the two faces of the ter of a typical one. In the higher concentration range protein layer, which tends to bend it towards the liq- dendritic patterns formed with stable tips. uid phase favoring dendritic growth. We propose that tip splitting and fractal growth instead of the formation of single crystals with 4-fold symmetry and stable tips D. Equilibrium shape can be attributed to the reduced anisotropy of the surface tension in case of the fast growth. Similarly, slowly The equilibrium shape of KCl crystals after the slow grownsinglecrystalcenteredstructuresareexplainedby drying method using 10 µl 5 mg/ml KCl with 5 mg/ml the decrease of anisotropy of surface tension during the FITC-BSAwasobservedbydropping5µlsaturatedKCl growth process. Our results demonstrate that the level solution on the crystals and covering the droplet with a ofanisotropycanchangedramaticallyduringthe growth 3 of a single pattern. This is likely to be caused by the unknown. Proteins can adhere to specific faces of the increase and the reaching of a critical level of protein crystal with increased affinity [2]. This anisotropic in- precipitationas the dropis drying up. Various examples teractioninfluences the anisotropyofthe surface tension of pattern formation driven by moving unstable inter- atthecrystal-proteininterfaceγc−p,whichmayresultin faces can be described by Laplaciangrowth. In this case the change of the direction of the fast growing tip. The the boundary condition along the Γ interface containing backgroundoftheproteinspecificbehaviorisunclear. As the dimensionless uΓ concentrationterm is [8, 9, 10, 11]: the experiment on the gold substrate indicates, in addi- tion to the protein-crystal interaction protein-substrate andcrystal-substrateeffectsalsoshouldbe consideredin uΓ =∆−d0(Θ)κ−β(Θ)vn, (3) further studies. where ∆is the undercooling,the capillarylengthd0 is proportional to the surface tension, κ denotes the local V. ACKNOWLEDGMENTS curvatureoftheinterface,β isthekineticcoefficient,Θis the angle between the normal to the surface and a fixed This work was supported by the Hungarian National crystallographic direction, v is the normal velocity of n Scientific Research Fund (OTKA, No. T-034995). We theinterface. Toexplainthestabletip-tipsplittingtran- thank Noémi Rozlosnik and Andra´s Czirók for the help sition,we proposethatd0 is alteredandits anisotropyis decreasedduetotheadherenceoftheproteinlayertothe FIG. 1: 80x80 µm2 deflection mode AFM image of tubulin KCl crystal surface. Other effects may also have an im- inducedpatternsofKClafterthefastdryingprocedure. Note pactonthemorphologyofKClpatterns. Thelowerdiffu- thetransition of theinitially 4-fold symmetriccrystallization sioncoefficientofK+andCl−ionsintheliquidphasedue to isotropic fractal growth at all the 4 tips of the structure tothehighproteinconcentrationduringdryingupmight caused byconsecutive tip splittings. induce transportlimited growth. Boththe decreasedve- locityoftheinterfaceandthemodificationoftheangular dependence of its coefficient (β) by a rather isotropic in- FIG. 2: 135x135 µm2 fluorescent image of 2 patterns of KCl corporation process of KCl into the crystal caused by crystals with different fractal dimensions grown in the pres- the protein coating can lead to lower anisotropy of the ence of FITC-BSA with the fast drying method. Brightness pattern. The incorporation of proteins into calcite crys- isproportional totheconcentration of FITC-BSA.Thecom- parisonwithphasecontrastandbrightfieldimagesprovethat tals is known to have an effect on the morphology [15]. albumin covers the crystals. However, fluorescent images displayed high protein concentration on the surface of crystals, we can not totally exclude incorporation. IgG typically and under some in establishing the instrumental background of our ex- conditions tubulin and albumin also induced the growth periments,andJuditOvádiandEmmaHlavandaforthe of elongated saw-toothed crystals instead of rectangular tubulin samples. needle-shaped ones. Branching saw-toothed structures werealsofound,theanglebetweenthebranchesis∼70◦. We argue that these are single crystals elongated in the (111) direction of the cubic crystal i.e., their axis lies FIG.3: 135x135µm2fluorescentimageofacubiccrystalcen- in this direction. The angle between (111) and direc- tered structure of KCl crystals induced by FITC-BSA using tions with the same symmetry e.g. (11-1) is 70.5◦. The theslowdryingmethod. Theanisotropicsinglecrystalinthe centeris surrounded by an isotropic pattern. mechanism leading to the formation of these crystals is [1] O. Antommattei, R. Heimer, D. G. Baker, H. R. Schu- macher, Jr., Clin. Exp.Rheum. 8, 29 (1990) FIG.4: 135x135 µm2 fluorescent image of stabletipped den- [2] D. Perl-Treves, L. Addadi, Proc. R. Soc. Lond. B 235, driticKClcrystalsinducedbyhighconcentrationofalbumin. 145 (1988) [3] E. Ortiz-Bravo, M. S. Sieck, H. R. Schumacher, Jr., Arthr.Rheum.36(9), 1274 (1993) [8] T. Vicsek, Fractal growth phenomena, 2nd ed., World [4] M.Kam,D.Perl-Treves,D.Caspi,L.Addadi,FASEBJ. Scientific, London, 1992, pp. 271-339. 6(8), 2608 (1992) [9] D. A. Kessler, J. Koplik, H. Levine, Adv. Phys. 37, 255 [5] M. Kam, D. Perl-Treves, R. Sfez, L. Addadi, J. Mol. (1988) Recognit. 7(4), 257 (1994) [10] E. Ben-Jacob, P. Garik, Nature 343, 523 (1990) [6] M.Yasui,M.Matsushita, J.Phys.Soc.Jap.61(7), 2327 [11] E. Ben-Jacob, N. Goldenfeld, J. S. Langer, G. Shön, (1992) Phys. Rev.Lett. 51(21), 1930 (1983) [7] J. Suda, M. Matsushita, J. Phys. Soc. Jap. 64(2), 348 [12] C. Nagl, E. Platzgummer, M. Schmid, P. Varga, S. (1995) Speller,W.Heiland,Phys.Rev.Lett.75(16),2976(1995) 4 [13] A-L. Barabási, Phys. Rev.Lett. 70(26), 4102 (1993) FIG. 5: 135x135 µm2 fluorescent image of KCl crystals with [14] G.C.Na,S.N.Timasheff,Biochemistry25,6214(1986) saw-toothed shape grown with IgG by the fast drying proce- [15] S. Albeck, I. Addadi, S. Weiner, Connect. Tissue Res. dure. The angle between the branches is ∼70◦. The bright 35(1-4), 365 (1996) contourofthecrystalscorrespondstotheIgG-FITCcoveron their surface. FIG.6: 205x205µm2fluorescentimage. Saw-toothedcrystals areshown inthecenterofapattern,whichisisotropic inthe surroundingregions. Thissamplewaspreparedwiththeslow FIG. 7: Morphological diagram of the protein induced pat- dryingprocedure in the presence of IgG. ternsof KCl crystals. This figure "fig1v.gif" is available in "gif"(cid:10) format from: http://arXiv.org/ps/physics/0209005v3 This figure "fig2v.jpg" is available in "jpg"(cid:10) format from: http://arXiv.org/ps/physics/0209005v3 This figure "fig3v.jpg" is available in "jpg"(cid:10) format from: http://arXiv.org/ps/physics/0209005v3 This figure "fig4v.jpg" is available in "jpg"(cid:10) format from: http://arXiv.org/ps/physics/0209005v3 This figure "fig5v.jpg" is available in "jpg"(cid:10) format from: http://arXiv.org/ps/physics/0209005v3 This figure "fig6v.jpg" is available in "jpg"(cid:10) format from: http://arXiv.org/ps/physics/0209005v3