Protein induced morphological transitions in KCl crystal growth

11 Pages·0.1 MB·English

by  Balint Szabo

Protein induced morphological transitions in KCl crystal growth
B´alint Szabo´ and Tama´s Vicsek
Department of Biological Physics, E¨otv¨os University,
Budapest, P´azm´any P´eter s´et´any 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, crys-
tallization, 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 sur-
face 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 No´emi Rozlosnik and Andra´s Czir´ok 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 con-
centration 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,andJuditOv´adiandEmmaHlavandaforthe
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
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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.
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