1090 DOI: 10.1021/la902464f Langmuir 2010, 26(2), 1090–1095Published on Web 10/30/2009

pubs.acs.org/Langmuir

© 2009 American Chemical Society

Nativeβ-Lactoglobulin Self-Assembles into aHexagonalColumnarPhase ona Solid Surface

Bruno Rizzuti,† Bruno Zappone,†,‡ Maria P. De Santo,†,‡ and Rita Guzzi*,‡,§

†Licryl CNR-INFMandCemif.Cal, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036Rende (CS), Italy,‡Department of Physics, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036Rende (CS), Italy, and §Unit�a

CNISM, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende (CS), Italy

Received July 8, 2009. Revised Manuscript Received September 11, 2009

Using electron scanning microscopy, we have studied the protein deposit left on silicon and mica substrates bydried droplets of aqueous solutions of bovine β-lactoglobulin at low concentration and pH= 2-7. We have observeddifferent self-assembled structures: homogeneous layers, hexagonal platelets and flower-shaped patterns laying flat onthe surface, and rods formed by columns. Homogeneous layers covered the largest area of the droplet deposit. The otherstructures were found in small isolated regions, where the protein solution dried in the form of microdroplets. Thepresence of hexagonal platelets, flower-shaped patterns and columnar rods shows that β-lactoglobulin self-assembles atthe surface in a hexagonal columnar phase, which has never been observed in solution. A comparison with proteinsshowing similar aggregates suggests that β-lactoglobulin structures grow from hexagonal germs composed of discoticnanometric building blocks, possibly possessing an octameric structure. We propose that discotic building blocks of β-lactoglobulin may be produced by the anisotropic interaction with the solid surface.

Introduction

Bovine β-lactoglobulin (β-LG) is the major whey protein ofcowmilk,1 with a concentration of about 2-3 g/L.2 It is one of themost studied biological molecules because of its interest in thefood industry for dairy products and processing,3 as well as inbasic biophysical research.4 Monomeric β-LG consists of aglobular polypeptide chain with 162 amino acid residues5,6 andthe protein is found predominantly as a dimer under the physio-logical conditions found in milk. β-LG occurs in several geneticvariants,7 the most abundant ones being A and B variants, whichdiffer from each other in only two residues, Asp64/Val118 andGly64/Ala118, respectively.

The equilibrium between monomers and dimers in aqueoussolution is mainly determined by the pH,8 but temperature,ionic strength and protein concentration also play a role.9-11

For pH<3.5, below the isoelectric point (pI = 5.112), β-LGmonomers are prevalent because of their mutual electrostatic

repulsion.13 An increase in the ionic strength decreases theintermolecular repulsion and induces the formation of dimers.10

For pH > 3.5 the protein forms dimers and the strength ofinteraction between monomers depends on the pH in a complexway.8

A fraction of the dimers can further associate in octamers,9,14-16

especially for variant A below room temperature and at pH =3.5-5.5. Aggregation in oligomers is also observed above theisoelectric point of β-LG and depends on temperature and proteinconcentration.11 These observations indicate that β-LG moleculesare able to self-associate in a variety of oligomers under differentchemico-physical conditions through electrostatic and other non-specific interactions.

While awealth of data is available for native β-LGoligomers insolution, information concerning larger aggregates is scarce.Most studies focus on β-LG structures resulting from heat-induced gelation and aggregation.3 Misfolded or partially un-foldedmolecules of β-LG are known to form a gel network that isprevalently composed of fine strands or particulate aggregates,depending whether the intermolecular electrostatic interaction isrepulsive or not, respectively.17 In contrast, native β-LG deposi-ted on mica and imaged with atomic force microscopy (AFM)produced a multilayer coverage with no indication of specificaggregates.18

In this work, we report on the large variety of structures thatwe observed in the deposit left by a dried droplet of dilutedsolution of native β-LG in the pH range 2-7 on silicon and micasurfaces imaged by environmental scanning electron microscope(ESEM). Our results indicate a self-assembly of the proteinmolecules into a hexagonal columnar phase as a result of theinteraction with the substrate. A comparison with proteins

*Corresponding author. Telephone:þ39.0984.496077. Fax:þ39.0984.494401.E-mail: guzzi@fis.unical.it.(1) Walstra, P.; Jenness, R. Dairy Chemistry and Physics; John Wiley & Sons:

New York, 1984.(2) Kontopidis, G.; Holt, C.; Sawyer, L. J. Dairy Sci. 2004, 87, 785–796.(3) Donald, A. M. Soft Matter 2008, 4, 1147–1150.(4) Sawyer, L.; Kontopidis, G. Biochim. Biophys. Acta 2000, 1482, 136–148.(5) Brownlow, S.; Morais Cabral, J. H.; Cooper, R.; Flower, D. R.; Yewdall,

S. J.; Polikarpov, I.; North, A. C. T.; Sawyer, L. Structure 1997, 5, 481–495.(6) Kuwata, K.; Hoshino, M.; Forge, V.; Era, S.; Batt, C. A.; Goto, Y. Protein

Sci. 1999, 8, 2541–2545.(7) Eigel, W. N.; Butler, J. E.; Ernstrom, C. A.; Farrel, H. M., Jr.; Harwalkar,

V. R.; Jenness, R.; Whitney, R. M. J. Dairy Sci. 1984, 67, 1599–1631.(8) Townend, R.;Winterbottom, R. J.; Timasheff, S. N. J. Am. Chem. Soc. 1960,

82, 3161–3168.(9) Townend, R.; Timasheff, S. N. J. Am. Chem. Soc. 1960, 82, 3168–3174.(10) Timasheff, S. N.; Townend, R. J. Am. Chem. Soc. 1961, 83, 470–473.(11) Verheul,M.; Pedersen, J. S.; Roefs, S. P. F.M.; deKruif, K.G.Biopolymers

1999, 49, 11–20.(12) Hambling, S. G.; McAlpine, A. S.; Sawyer, L. In Advanced Dairy Chem-

istry; Fox, P., Ed.; Elsevier Applied Science Publishers Ltd.: London, 1992; Vol. 1, pp141-190.(13) Townend, R.; Weinberger, L.; Timasheff, S. N. J. Am. Chem. Soc. 1960, 82,

3175–3179.

(14) Witz, J.; Timasheff, S. N.; Luzzati, V. J. Am. Chem. Soc. 1964, 86, 168–173.(15) Kumosinski, T. F.; Timasheff, S. N. J. Am. Chem. Soc. 1966, 88, 5635–5642.(16) McKenzie, H. A.; Sawyer, W. H.; Smith, M. B. Biochim. Biophys. Acta

1967, 147, 73–92.(17) Lef�evre, T.; Subirade, M. Biopolymers 2000, 54, 578–586.(18) Ikeda, S.; Morris, V. J. Biomacromolecules 2002, 3, 382–389.

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showing similar aggregation suggests that the molecular buildingblock of the columnar hexagonal phase could be a discoticoligomer, possibly an octamer.

Materials and Methods

Lyophilized β-LG from bovine milk (purity 90%) was pur-chased from Sigma-Aldrich (Poole, U.K., lot no. 052K7017) andused as received. Protein powderwas dissolved in bidistilled waterat a concentration of 20 mg/mL, stirred for 60 min, and thendiluted to 0.4 μg/mL. The pH was adjusted at the value indicated(from 2 to 7) by adding small amounts of hydrochloric acid.

In some experiments, the protein solution was filtered throughplastic filters with 0.2 μm pores. Aliquots (2 μL) of the dilutedsample solutions were deposited on the substrates in a clean roomand left drying in air at room temperature. We used surfaces ofeither freshly cleaved mica or silicon cleaned in piranha solution(sulfuric acid and hydrogen peroxide 3:1 v/v).

Environmental scanning electron microscope (ESEM, QuantaFEG 400 by FEI) imaging was done within 24 h from sampledeposition. The chamber pressure was 1 mbar, and the accelera-tion voltage was 5-10 kV.

Results

Parts a-c of Figure 1 showESEM images of the protein depositleft by a droplet of β-LG solution on silicon at different pH,respectively, 6.5, 5, and 2. The images show regions of uniformcolor, alternatively bright or dark, covering areas of hundredsof μm2. A uniform brightness in ESEM images indicates aregion with uniform thickness. Such homogeneous layers withlarge holes were found on all samples in various regions of theentire droplet deposit and on the whole range of pH investigated,between 2 and 7. Figure 1d shows a similar layer coverageobtained on mica under at pH = 5. Note that images obtainedon mica generally have a lower quality than on silicon, because

mica is nonconductive and becomes charged during scanningof the ESEM beam, rapidly becoming reflective to incomingelectrons.

Figure 2a shows protein platelets found on silicon at neutralpH, in coexistence with the layer coating. The platelets wereapproximately hexagonal with edges forming angles close to 120�(Figure 2b). Some of these aggregates, such as the one shown inFigure 2b, are formedbya cluster of platelets butmost of themareconstituted by single isolated hexagons. These isolated plateletshave a size of 0.2( 0.1 μm,whereas the size of clusters is 0.8( 0.5μm. Exceptionally, single hexagons with bigger dimension (1 μm)have been found, only within clusters. In a cluster are found onaverage 5 platelets. In regions containing platelets/clusters, thesestructure are uniformly distributed (1 structure per 10 μm2).Hexagonal platelets were also observed on silicon for pH = 2,well below the isoelectric point ofβ-LG (Figure 2c), although theyare less common.

Microdroplets of proteinaceous material were occasionallyfound on mica at pH = 5 and appeared as dark domains, asshown in Figure 3a. Within these microdroplets the proteinformed platelets appearing bright in the ESEM images. Thisindicates that these aggregates have a different structure withrespect to the amorphous protein deposit, probablymore compactand ordered. These aggregates appear to grow from and inside theamorphous protein deposit. Most aggregates had straight edges,often forming an angle close to 120�; however, these aggregates donot have a hexagonal shape, but rather a “flower” outline(Figure 3b). Many of these structures appeared nibbled andshowed dark inner regions, which could be empty or amorphousregions. The size of the aggregates was 0.12 ( 0.03 μm.

Amorphous microdroplets were found on silicon more fre-quently and with bigger size than on mica. Figure 4a shows aprotein deposit left on silicon at pH=2. In someof these domains

Figure 1. Homogeneous protein layer on silicon at (a) pH= 6.5, (b) pH= 5, and (c) pH= 2 and (d) on mica at pH= 2. Bright and darkareas correspond to regions with different heights, likely a single layer with holes exposing the underlying surface.

1092 DOI: 10.1021/la902464f Langmuir 2010, 26(2), 1090–1095

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Figure 2. Hexagonal crystals of β-LGon silicon at low and neutral pH. (a) Large area scan for pH=7. (b)Detail of part a showing a clusterof crystals. (c) Isolated crystal at pH= 2. Dashed lines indicate aggregates with straight edges forming an angle close to 120�.

Figure 3. (a) Flower-shaped structures contained in a drop-like domain on mica at pH= 5. (b) Detail of part a. Dashed lines in the insetindicate aggregates with straight edges forming an angle close to 120�.

Figure 4. (a) Flower-shaped structures contained in a drop-like domain on silicon at pH = 2. The elongated structures at the bottom leftcorner of the image are shown in better details in Figure 5. (b) Detail of part a.

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the protein formed aggregates with a flower-shaped pattern and asize of 0.9( 0.3 μm, as shown inFigure 4b. Compared to plateletsobserved on mica, flower-shaped aggregates on silicon appear togrowhigherwithin the amorphousmatrix, as can be inferred fromthe differences of brightness of the image within the sameaggregate.

On silicon, microdroplets with diameters comprised between1 and 20 μm contain more complex aggregates (Figure 4a bottomleft corner, and Figure 5). As shown in Figure 5b, the centralregion ofmost of suchmicrodroplets contains rod-like aggregatesand crenulated flower-shaped patterns, often in coexistence(Figure 5b). Rod-like aggregates appeared as bunches of elon-gated rods, with a tendency toward a bilateral symmetry.Figure 5a shows that rod size increased with the diameter of themicrodroplet, whereas the length-to-width aspect ratio remainedapproximately constant. Droplets with a diameter smaller thanabout 1 μm did not contain any rod. ESEM images were oftenblurred due to the difficulty in bringing different parts of thestructure in the same focus, indicating that rods are significantlythicker than the other structures. In large microdroplets, rodscould expand their ends in the third dimension and acquire abone-shaped aspect; in such cases, ESEM images (not shown)were completely blurred.

Finally, we point out that the structures described above werealso observed for protein solutions that were filtered beforedeposition. Therefore, the structures are not formed in solution,but they result from the interaction with the substrate.

Discussion

Native β-LG is a protein that can self-assemble in a variety ofways in solution.8,9,11,19 In contrast, little is known about β-LGaggregation on a surface. By using ESEM microscopy, we haveobserved a variety of aggregation structures: homogeneous layers(Figure 1), hexagonal platelets (Figure 2 and 3), flower-shapedpatterns (Figures 3-5) and elongated rods (Figure 5).

The homogeneous layers are observed on silicon and mica forall experimental conditions considered. Similar layers have beenpreviously found by AFM for β-LG absorbed on mica at neutralpH18 and interpreted as monolayers or multilayers in directcontact with the substrate. Most likely, the homogeneous layersshown in Figure 1 are similar structures due to direct adsorptionof the protein on the surface, probably favored by the amphiphiliccharacter of adsorbed molecules.20 The presence of compact

regions and large holes, instead of a continuous coverage,indicates that in addition to protein-surface interaction there isa lateral protein-protein interaction in the layer.

Hexagonal platelets (Figure 2) and flower-shaped patterns(Figures 3 and 4) are more complex structures that indicateprotein self-assembly into a hexagonal phase. Most likely, hexa-gonal plates are single crystals resulting from slow growth close toequilibrium conditions, whereas flower-shaped patterns appearunder faster growth conditions that favor branching from thehexagonal crystal vertices.21Flower-shaped aggregates are alwaysfound inside microdroplets in an amorphous material that ap-pears as a dark background in Figures 3-5.

Elongated rods grow in a similar environment as hexagonalplatelets and flower-shaped patterns and often coexist with them(Figure 5b). Rods appear to grow approximately parallel to thesurface but they show a more three-dimensional character thanthe former structures, indicating that rods are mainly due toprotein-protein, rather than protein-substrate interaction.Figure 5b shows flower-shaped patterns with a bumpy surfaceat the edge of a microdroplet which contains a bunch of columnsat its center. This suggests that rods may grow platelets only inregions where the droplet is sufficiently thick.

A tendency of bovine β-LG to form hexagonal aggregates isnot entirely unexpected. Flat hexagonal plates were previouslygrown in an attempt to determine the protein crystal structure atlow pH.2,22 However, our results are significant because theyclearly indicate that this behavior is spontaneous in the presenceof a surface under close-to-native conditions.

The formation of hexagonal aggregates is not uncommon inproteinaceous precipitants and has already been reported inseveral cases. For instance, capsomers of large icosahedralviruses23 and capsid-like protein shells (such as those obtainedby the major carboxysome shell proteins)24,25 show flat hexa-gons upon crystallization and ATP synthase can form26 (orinduce other proteins to form)27 hexagonal ring-like structures.

Figure 5. (a) Elongated protein aggregates contained in drop-like domains on silicon at pH = 2. (b) Detail of part a showing the multi-columnar nature of the elongated structures in coexistence with flower-shaped aggregates.

(19) Piazza, R.; Iacopini, S. Eur. Phys. J. E 2002, 7, 45–48.(20) Cicuta, P. J. Colloid Interface Sci. 2007, 308, 93–99.

(21) Bouligand, Y. J. Phys. (Paris) 1980, 41, 1307–1315.(22) Aschaffenburg, R.; Green, D. W.; Simmons, R. M. J. Mol. Biol. 1965, 13,

194–201.(23) Kuznetsov, Yu. G.; Malkin, A. J.; Lucas, R. W.; McPherson, A. Colloids

Surf., B 2000, 19, 333–346.(24) Yeates, T. O.; Tsai, Y.; Tanaka, S.; Sawaya,M. R.; Kerfeld, C. A. Biochem.

Soc. T. 2007, 35, 508–511.(25) Tsai, Y.; Sawaya, M. R.; Cannon, G. C.; Cai, F.; Williams, E. B.;

Heinhorst, S.; Kerfeld, C. A.; Yeates, T. O. PLoS Biol. 2007, 5, e144.(26) Boekema, E. J.; van Heel, M.; Gr€aber, P. Biochim. Biophys. Acta 1988, 933,

365–371.(27) Hayashi, F.; Suzuki, H.; Iwase, R.; Uzumaki, T.; Miyake, A.; Shen, J.-R.;

Imada, K.; Furukawa, Y.; Yonekura, K.; Namba, K.; Ishiura, M. Genes Cells2003, 8, 287–296.

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However, except for their hexagonal symmetry, such structuresdo not share other similarities with those obtained for β-LG.

A general survey in search for proteins forming hexagonalplatelets, performed at the best of our ability, revealed that onlythree proteins showing a hexagonal phase share other similaritieswith β-LG, for instance the growth of hexagonal structures insidean amorphous matrix (Figures 3 and 4) or the formation of rods(Figure 5).

Lobster carapace carotenoprotein R-crustacyanin belongs tothe same protein family as β-LG, namely lipocalins, and formssmall hexagonal plates or columns in the presence of differentsalts, as well as more complex structures that include largehexagonal rods and needles under a variety of experimentalconditions.28

Another protein producing similar aggregates is pot0434017(also known as FL11) from Pyrococcus sp. OT3, a full-lengthfeast/famine regulatory protein29 showing hexagonal assembliesgrownwithin an amorphousmatrix ona surface. Images obtainedby cryo-electron microscopy30 reveal structures that are inter-mediate between hexagonal crystals and unstructured aggregates,very similar to the ones that are obtained for β-LG (Figure 2b).For pot0434017, it has been suggested that germinal crystals canform and grow inside amorphous precipitants by rearrangingthe protein molecules,30 a mechanism that could be invoked forβ-LG, too. Pot0434017 also forms unbranched columnar struc-tures (tubes) in thin films.30

Hexagonal structures similar to those found for β-LG are alsoobserved for nucleosome core particles (NCPs), the protein/DNAcomplex that constitutes the fundamental repeating units ofeukaryotic chromatin.31,32 In fact, a discotic columnarmesophaseis obtained by slow dehydration of a concentrated solution in thepresence of high amounts of monovalent salts favoring particleaggregation.33,34 Regular hexagons are observed in preparationsstabilized for a long time, ranging from days to months. Inter-estingly, NCP solutions confined in thin samples (5-10 μm),where growth occurs in quasi two-dimensional conditions, lead totwo distinct conformations for the protein aggregates: elongatedrods with possible dendritic shape and hexagonal platelets evol-ving into characteristic flower-shaped patterns.33

These structures have been explained in terms of self-orderingof the discotic NCP building blocks organized into columns,33 agrowth that is typical of discotic hexagonal germs.21 We believethat a similar mechanism determines the structures observed forβ-LG (i.e., hexagonal platelets, flower-shaped patterns, andelongated rods), which can be explained in terms of growth of ahexagonal columnar phase starting from flat crystalline germs atthe surface, as illustrated in Figure 6. In the presence of a surface,when the axis of the column is normal to the plane, the growthoccurs along the radial direction and leads to hexagonal crystalsand flower-shaped patterns. Growth of extended structures in thedirection normal to the surface is hindered by the limited thick-ness of the sample. When the columns are aligned parallel to thesurface, they can freely elongate, and they adhere to each otherforming rods.

One of the few differences between β-LGandNCP structures isthat the flower-shaped patterns obtained for β-LG are often far

from having a perfect 6-fold symmetry. This can be due to thefact that the growth for β-LG structures is much faster, since thedrop of solution dries within a fewminutes after its deposition onthe substrate. β-LG rods never show the dendritic appearancereported for NCPs: β-LG rods radiate from a single region buteach rod is composed by roughly parallel columns (Figure 5b).This dissimilarity could be due to the chiral nature of NCPs,leading to a twist among the columns that facilitates dendriticbranching.34

Analogies among β-LG, R-crustacyanin, pot0434017, andNCP, are striking, given their differences in sequence and molec-ular structure. Comparison among these proteins gives importantindications on themolecular scalemechanisms of self-assembly ofsingle β-LG monomers into aggregates on the microscopic scale.Although no crystal structure exists for native R-crustacyanin,several biophysical and biochemical studies indicate that it formsan octamer of heterodimers.35 Pot0434017 is predominantlydimeric in solution and is believed to form discotic octamers withfour dimers.36 NCP consists in a histone octamer with approxi-mately 146 base pairs of superhelical DNA wrapped around it.37

Therefore, it could be hypothesized that also β-LG self-assemblesat the surface in a discotic form, possibly an octamer, providedthat the protein concentration is sufficiently high to form seeds.Otherwise, if the local concentration is not sufficient, β-LGdeposits as a monolayer as previously reported.18

Experimental conditions such as the surface type and the pHare important in determining aggregation, although it is not easyto identify their specific role. On silicon, flat hexagonal plateletsand flower-shaped structures are observed for all pH values,whereas elongated rods are observed only at low pH. On mica,formation of the hexagonal phase is suggested by the presence offlower-shaped patterns at pH=5.Anumber of reasons can hinderthe formation of defined hexagonal structures on mica. First ofall, mica is more hydrophilic than silicon. Drops of equal volumeof protein solution wet mica surfaces, whereas they do not spreadon silicon substrates.Water evaporation is faster onmica becauseit takes place on a larger surface area, therefore protein structuresgrow faster and are less regular than on silicon. Second, since thesolution spreads over a large surface area, the protein distributesmore uniformly and cannot reach a high concentration on local,

Figure 6. Schematic representation of the growth of hexagonaland columnar structures ofβ-LG. In solution, the largemajority ofproteinmolecules aremonomers or dimers. (a)When deposited ona solid surface, molecules self-assemble into discotic oligomers.The average orientation of such oligomers (the director n) can beeither (b) perpendicular or (c) parallel to the surface. In the formercase, hexagonal germs are formed that may evolve to flower-shaped platelets, whereas in the latter case columns are formedwhich can elongate and bunch together in rods.

(28) Flower, D. R. Biochim. Biophys. Acta 2000, 1482, 46–56.(29) Koike, H.; Ishijima, S. A.; Clowney, L.; Suzuki, M. Proc. Natl. Acad. Sci.

U.S.A. 2004, 101, 2840–2845.(30) Ishijima, S. A.; Clowney, L.; Koike, H.; Suzuki, M. Proc. Jpn. Acad. 2004,

80B, 22–27.(31) Olins, A. L.; Olins, D. E. Science 1974, 183, 330–332.(32) Kornberg, R. D. Science 1974, 184, 868–871.(33) Leforestier, A.; Livolant, F. Biophys. J. 1997, 73, 1771–1776.(34) Livolant, F.; Leforestier, A. Biophys. J. 2000, 78, 2716–2729.

(35) Zagalsky, P. F. In Carotenoids; Britton, G., Liaaen-Jensen, S., Pfander, H.,Eds.; Birkh€auser: Basel, 1995; Vol. 1A, pp 287-294.

(36) Koike, H.; Sakuma, M.; Mikami, A.; Amano, N.; Suzuki, M. Proc. Jpn.Acad. 2003, 79B, 63–69.

(37) Luger, K.; M€ader, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T.J. Nature 1997, 389, 251–260.

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isolatedmicrodroplets. This condition canpromote the formationof a protein layer (such as in Figure 1d), hamper the developmentof flower-shaped patterns (Figure 3) and inhibit the formation ofelongated rods, never observed on mica. Finally, the surface ofmica is negatively charged and this can interfere with theaggregation of β-LG. In fact, flower-shaped structures are onlyobserved on mica at pH= 5, close to the isoelectric point of theprotein, that is the condition that favors aggregation the most.12

Moreover, the low quality of ESEM images on mica may have insome cases prevented the detection of aggregates.

Finally, we would like to point out that there is a certain degreeof non reproducibility in our experiments. The formation ofmicroscopic amorphous regions with an appropriate proteinconcentration depends on the complex process of drying of adroplet. Moreover, the formation of suitable aggregation seeds isan essentially random process. It is possible that small casualdifferences in the experimental conditions can influence thisprocess. In particular, the growth of the structures appears tobe affected by the local thickness of the protein deposit. Slowgrowth in thin deposit (for example around the edge of amicrodroplet, Figure 5a) seems to favor flat aggregates, i.e.,hexagonal crystals and flower-shaped patterns. Large rod-likestructures are only observed on silicon at the center of micro-droplets with a diameterg1 μm. In very large droplets, columnar

bunches have room to elongate and diverge, adopting a bone-shaped structure and other less regular forms.

Conclusion

Avariety of protein structures have been observed for native β-LG deposited on a surface. Hexagonal plates, flower-shapedpatterns and elongated rods indicate that the protein self-assem-bles into a hexagonal columnar phase. We suggest that theanisotropic interaction with the surface leads to the formationof discotic oligomers andwe hypothesize anoctameric quaternarystructure for these molecular building blocks. The degree ofhydrophilicity of the substrate and the pH of the deposited β-LG solution are both important to determine the self-organiza-tion of the protein. In particular, the most complex structures areobserved onpartially hydrophobic substrate (silicon) at pHbelowthe protein isoelectric point. Further studies will be required tounderstand the influence of these and other physicochemicalparameters relevant to aggregation, such as the protein concen-tration, ionic strength, temperature, and dehydration conditions.

Acknowledgment.We thankDr.GiovanniDesiderio of LicrylCNR-INFMfor helpful technical assistancewith theESEM.Thiswork was partially funded by CNR-INFM (Project MD.P01.021“Bio-soft matter and Health Science”).