Native β-Lactoglobulin Self-Assembles into a Hexagonal Columnar

Oct 30, 2009 - ‡Department of Physics, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende (CS), Italy, and §Unit`a. CNISM, University of...
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Native β-Lactoglobulin Self-Assembles into a Hexagonal Columnar Phase on a Solid Surface Bruno Rizzuti,† Bruno Zappone,†,‡ Maria P. De Santo,†,‡ and Rita Guzzi*,‡,§ † ‡

Licryl CNR-INFM and Cemif.Cal, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende (CS), Italy, Department of Physics, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende (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 by dried droplets of aqueous solutions of bovine β-lactoglobulin at low concentration and pH = 2-7. We have observed different self-assembled structures: homogeneous layers, hexagonal platelets and flower-shaped patterns laying flat on the surface, and rods formed by columns. Homogeneous layers covered the largest area of the droplet deposit. The other structures were found in small isolated regions, where the protein solution dried in the form of microdroplets. The presence of hexagonal platelets, flower-shaped patterns and columnar rods shows that β-lactoglobulin self-assembles at the surface in a hexagonal columnar phase, which has never been observed in solution. A comparison with proteins showing similar aggregates suggests that β-lactoglobulin structures grow from hexagonal germs composed of discotic nanometric building blocks, possibly possessing an octameric structure. We propose that discotic building blocks of βlactoglobulin may be produced by the anisotropic interaction with the solid surface.

Introduction Bovine β-lactoglobulin (β-LG) is the major whey protein of cow milk,1 with a concentration of about 2-3 g/L.2 It is one of the most studied biological molecules because of its interest in the food industry for dairy products and processing,3 as well as in basic biophysical research.4 Monomeric β-LG consists of a globular polypeptide chain with 162 amino acid residues5,6 and the protein is found predominantly as a dimer under the physiological conditions found in milk. β-LG occurs in several genetic variants,7 the most abundant ones being A and B variants, which differ from each other in only two residues, Asp64/Val118 and Gly64/Ala118, respectively. The equilibrium between monomers and dimers in aqueous solution 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-LG monomers are prevalent because of their mutual electrostatic *Corresponding author. Telephone: þ39.0984.496077. Fax: þ39.0984.494401. E-mail: [email protected]. (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.; de Kruif, K. G. Biopolymers 1999, 49, 11–20. (12) Hambling, S. G.; McAlpine, A. S.; Sawyer, L. In Advanced Dairy Chemistry; Fox, P., Ed.; Elsevier Applied Science Publishers Ltd.: London, 1992; Vol. 1, pp 141-190. (13) Townend, R.; Weinberger, L.; Timasheff, S. N. J. Am. Chem. Soc. 1960, 82, 3175–3179.

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repulsion.13 An increase in the ionic strength decreases the intermolecular repulsion and induces the formation of dimers.10 For pH > 3.5 the protein forms dimers and the strength of interaction between monomers depends on the pH in a complex way.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 the isoelectric point of β-LG and depends on temperature and protein concentration.11 These observations indicate that β-LG molecules are able to self-associate in a variety of oligomers under different chemico-physical conditions through electrostatic and other nonspecific interactions. While a wealth of data is available for native β-LG oligomers in solution, information concerning larger aggregates is scarce. Most studies focus on β-LG structures resulting from heatinduced gelation and aggregation.3 Misfolded or partially unfolded molecules of β-LG are known to form a gel network that is prevalently composed of fine strands or particulate aggregates, depending whether the intermolecular electrostatic interaction is repulsive or not, respectively.17 In contrast, native β-LG deposited on mica and imaged with atomic force microscopy (AFM) produced a multilayer coverage with no indication of specific aggregates.18 In this work, we report on the large variety of structures that we observed in the deposit left by a dried droplet of diluted solution of native β-LG in the pH range 2-7 on silicon and mica surfaces imaged by environmental scanning electron microscope (ESEM). Our results indicate a self-assembly of the protein molecules into a hexagonal columnar phase as a result of the interaction with the substrate. A comparison with proteins (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) Lefevre, T.; Subirade, M. Biopolymers 2000, 54, 578–586. (18) Ikeda, S.; Morris, V. J. Biomacromolecules 2002, 3, 382–389.

Published on Web 10/30/2009

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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 dark areas correspond to regions with different heights, likely a single layer with holes exposing the underlying surface.

showing similar aggregation suggests that the molecular building block of the columnar hexagonal phase could be a discotic oligomer, possibly an octamer.

Materials and Methods Lyophilized β-LG from bovine milk (purity 90%) was purchased from Sigma-Aldrich (Poole, U.K., lot no. 052K7017) and used as received. Protein powder was dissolved in bidistilled water at a concentration of 20 mg/mL, stirred for 60 min, and then diluted 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 through plastic filters with 0.2 μm pores. Aliquots (2 μL) of the diluted sample solutions were deposited on the substrates in a clean room and left drying in air at room temperature. We used surfaces of either freshly cleaved mica or silicon cleaned in piranha solution (sulfuric acid and hydrogen peroxide 3:1 v/v). Environmental scanning electron microscope (ESEM, Quanta FEG 400 by FEI) imaging was done within 24 h from sample deposition. The chamber pressure was 1 mbar, and the acceleration voltage was 5-10 kV.

Results Parts a-c of Figure 1 show ESEM images of the protein deposit left by a droplet of β-LG solution on silicon at different pH, respectively, 6.5, 5, and 2. The images show regions of uniform color, alternatively bright or dark, covering areas of hundreds of μm2. A uniform brightness in ESEM images indicates a region with uniform thickness. Such homogeneous layers with large holes were found on all samples in various regions of the entire droplet deposit and on the whole range of pH investigated, between 2 and 7. Figure 1d shows a similar layer coverage obtained on mica under at pH = 5. Note that images obtained on mica generally have a lower quality than on silicon, because Langmuir 2010, 26(2), 1090–1095

mica is nonconductive and becomes charged during scanning of the ESEM beam, rapidly becoming reflective to incoming electrons. Figure 2a shows protein platelets found on silicon at neutral pH, in coexistence with the layer coating. The platelets were approximately hexagonal with edges forming angles close to 120° (Figure 2b). Some of these aggregates, such as the one shown in Figure 2b, are formed by a cluster of platelets but most of them are constituted by single isolated hexagons. These isolated platelets have a size of 0.2 ( 0.1 μm, whereas the size of clusters is 0.8 ( 0.5 μm. Exceptionally, single hexagons with bigger dimension (1 μm) have been found, only within clusters. In a cluster are found on average 5 platelets. In regions containing platelets/clusters, these structure are uniformly distributed (1 structure per 10 μm2). Hexagonal platelets were also observed on silicon for pH = 2, well below the isoelectric point of β-LG (Figure 2c), although they are less common. Microdroplets of proteinaceous material were occasionally found on mica at pH = 5 and appeared as dark domains, as shown in Figure 3a. Within these microdroplets the protein formed platelets appearing bright in the ESEM images. This indicates that these aggregates have a different structure with respect to the amorphous protein deposit, probably more compact and ordered. These aggregates appear to grow from and inside the amorphous protein deposit. Most aggregates had straight edges, often forming an angle close to 120°; however, these aggregates do not have a hexagonal shape, but rather a “flower” outline (Figure 3b). Many of these structures appeared nibbled and showed dark inner regions, which could be empty or amorphous regions. The size of the aggregates was 0.12 ( 0.03 μm. Amorphous microdroplets were found on silicon more frequently and with bigger size than on mica. Figure 4a shows a protein deposit left on silicon at pH = 2. In some of these domains DOI: 10.1021/la902464f

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Figure 2. Hexagonal crystals of β-LG on silicon at low and neutral pH. (a) Large area scan for pH = 7. (b) Detail of part a showing a cluster of 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 inset indicate 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 left corner of the image are shown in better details in Figure 5. (b) Detail of part a. 1092 DOI: 10.1021/la902464f

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

the protein formed aggregates with a flower-shaped pattern and a size of 0.9 ( 0.3 μm, as shown in Figure 4b. Compared to platelets observed on mica, flower-shaped aggregates on silicon appear to grow higher within the amorphous matrix, as can be inferred from the differences of brightness of the image within the same aggregate. On silicon, microdroplets with diameters comprised between 1 and 20 μm contain more complex aggregates (Figure 4a bottom left corner, and Figure 5). As shown in Figure 5b, the central region of most of such microdroplets contains rod-like aggregates and crenulated flower-shaped patterns, often in coexistence (Figure 5b). Rod-like aggregates appeared as bunches of elongated rods, with a tendency toward a bilateral symmetry. Figure 5a shows that rod size increased with the diameter of the microdroplet, whereas the length-to-width aspect ratio remained approximately constant. Droplets with a diameter smaller than about 1 μm did not contain any rod. ESEM images were often blurred due to the difficulty in bringing different parts of the structure in the same focus, indicating that rods are significantly thicker than the other structures. In large microdroplets, rods could expand their ends in the third dimension and acquire a bone-shaped aspect; in such cases, ESEM images (not shown) were completely blurred. Finally, we point out that the structures described above were also observed for protein solutions that were filtered before deposition. 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 of ways in solution.8,9,11,19 In contrast, little is known about β-LG aggregation on a surface. By using ESEM microscopy, we have observed a variety of aggregation structures: homogeneous layers (Figure 1), hexagonal platelets (Figure 2 and 3), flower-shaped patterns (Figures 3-5) and elongated rods (Figure 5). The homogeneous layers are observed on silicon and mica for all experimental conditions considered. Similar layers have been previously found by AFM for β-LG absorbed on mica at neutral pH18 and interpreted as monolayers or multilayers in direct contact with the substrate. Most likely, the homogeneous layers shown in Figure 1 are similar structures due to direct adsorption of the protein on the surface, probably favored by the amphiphilic character of adsorbed molecules.20 The presence of compact (19) Piazza, R.; Iacopini, S. Eur. Phys. J. E 2002, 7, 45–48. (20) Cicuta, P. J. Colloid Interface Sci. 2007, 308, 93–99.

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regions and large holes, instead of a continuous coverage, indicates that in addition to protein-surface interaction there is a lateral protein-protein interaction in the layer. Hexagonal platelets (Figure 2) and flower-shaped patterns (Figures 3 and 4) are more complex structures that indicate protein self-assembly into a hexagonal phase. Most likely, hexagonal plates are single crystals resulting from slow growth close to equilibrium conditions, whereas flower-shaped patterns appear under faster growth conditions that favor branching from the hexagonal crystal vertices.21 Flower-shaped aggregates are always found inside microdroplets in an amorphous material that appears as a dark background in Figures 3-5. Elongated rods grow in a similar environment as hexagonal platelets and flower-shaped patterns and often coexist with them (Figure 5b). Rods appear to grow approximately parallel to the surface but they show a more three-dimensional character than the former structures, indicating that rods are mainly due to protein-protein, rather than protein-substrate interaction. Figure 5b shows flower-shaped patterns with a bumpy surface at the edge of a microdroplet which contains a bunch of columns at its center. This suggests that rods may grow platelets only in regions where the droplet is sufficiently thick. A tendency of bovine β-LG to form hexagonal aggregates is not entirely unexpected. Flat hexagonal plates were previously grown in an attempt to determine the protein crystal structure at low pH.2,22 However, our results are significant because they clearly indicate that this behavior is spontaneous in the presence of a surface under close-to-native conditions. The formation of hexagonal aggregates is not uncommon in proteinaceous precipitants and has already been reported in several cases. For instance, capsomers of large icosahedral viruses23 and capsid-like protein shells (such as those obtained by the major carboxysome shell proteins)24,25 show flat hexagons upon crystallization and ATP synthase can form26 (or induce other proteins to form)27 hexagonal ring-like structures. (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 Cells 2003, 8, 287–296.

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However, except for their hexagonal symmetry, such structures do not share other similarities with those obtained for β-LG. A general survey in search for proteins forming hexagonal platelets, performed at the best of our ability, revealed that only three proteins showing a hexagonal phase share other similarities with β-LG, for instance the growth of hexagonal structures inside an amorphous matrix (Figures 3 and 4) or the formation of rods (Figure 5). Lobster carapace carotenoprotein R-crustacyanin belongs to the same protein family as β-LG, namely lipocalins, and forms small hexagonal plates or columns in the presence of different salts, as well as more complex structures that include large hexagonal rods and needles under a variety of experimental conditions.28 Another protein producing similar aggregates is pot0434017 (also known as FL11) from Pyrococcus sp. OT3, a full-length feast/famine regulatory protein29 showing hexagonal assemblies grown within an amorphous matrix on a surface. Images obtained by cryo-electron microscopy30 reveal structures that are intermediate 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 can form and grow inside amorphous precipitants by rearranging the protein molecules,30 a mechanism that could be invoked for β-LG, too. Pot0434017 also forms unbranched columnar structures (tubes) in thin films.30 Hexagonal structures similar to those found for β-LG are also observed for nucleosome core particles (NCPs), the protein/DNA complex that constitutes the fundamental repeating units of eukaryotic chromatin.31,32 In fact, a discotic columnar mesophase is obtained by slow dehydration of a concentrated solution in the presence of high amounts of monovalent salts favoring particle aggregation.33,34 Regular hexagons are observed in preparations stabilized for a long time, ranging from days to months. Interestingly, NCP solutions confined in thin samples (5-10 μm), where growth occurs in quasi two-dimensional conditions, lead to two distinct conformations for the protein aggregates: elongated rods with possible dendritic shape and hexagonal platelets evolving into characteristic flower-shaped patterns.33 These structures have been explained in terms of self-ordering of the discotic NCP building blocks organized into columns,33 a growth that is typical of discotic hexagonal germs.21 We believe that a similar mechanism determines the structures observed for β-LG (i.e., hexagonal platelets, flower-shaped patterns, and elongated rods), which can be explained in terms of growth of a hexagonal columnar phase starting from flat crystalline germs at the surface, as illustrated in Figure 6. In the presence of a surface, when the axis of the column is normal to the plane, the growth occurs along the radial direction and leads to hexagonal crystals and flower-shaped patterns. Growth of extended structures in the direction normal to the surface is hindered by the limited thickness of the sample. When the columns are aligned parallel to the surface, they can freely elongate, and they adhere to each other forming rods. One of the few differences between β-LG and NCP structures is that the flower-shaped patterns obtained for β-LG are often far (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.

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Figure 6. Schematic representation of the growth of hexagonal and columnar structures of β-LG. In solution, the large majority of protein molecules are monomers or dimers. (a) When deposited on a solid surface, molecules self-assemble into discotic oligomers. The average orientation of such oligomers (the director n) can be either (b) perpendicular or (c) parallel to the surface. In the former case, hexagonal germs are formed that may evolve to flowershaped platelets, whereas in the latter case columns are formed which can elongate and bunch together in rods.

from having a perfect 6-fold symmetry. This can be due to the fact that the growth for β-LG structures is much faster, since the drop of solution dries within a few minutes after its deposition on the substrate. β-LG rods never show the dendritic appearance reported for NCPs: β-LG rods radiate from a single region but each 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 dendritic branching.34 Analogies among β-LG, R-crustacyanin, pot0434017, and NCP, are striking, given their differences in sequence and molecular structure. Comparison among these proteins gives important indications on the molecular scale mechanisms of self-assembly of single 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 forms an octamer of heterodimers.35 Pot0434017 is predominantly dimeric in solution and is believed to form discotic octamers with four dimers.36 NCP consists in a histone octamer with approximately 146 base pairs of superhelical DNA wrapped around it.37 Therefore, it could be hypothesized that also β-LG self-assembles at the surface in a discotic form, possibly an octamer, provided that the protein concentration is sufficiently high to form seeds. Otherwise, if the local concentration is not sufficient, β-LG deposits as a monolayer as previously reported.18 Experimental conditions such as the surface type and the pH are important in determining aggregation, although it is not easy to identify their specific role. On silicon, flat hexagonal platelets and 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 of flower-shaped patterns at pH=5. A number of reasons can hinder the formation of defined hexagonal structures on mica. First of all, mica is more hydrophilic than silicon. Drops of equal volume of protein solution wet mica surfaces, whereas they do not spread on silicon substrates. Water evaporation is faster on mica because it takes place on a larger surface area, therefore protein structures grow faster and are less regular than on silicon. Second, since the solution spreads over a large surface area, the protein distributes more uniformly and cannot reach a high concentration on local, (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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isolated microdroplets. This condition can promote the formation of a protein layer (such as in Figure 1d), hamper the development of flower-shaped patterns (Figure 3) and inhibit the formation of elongated rods, never observed on mica. Finally, the surface of mica is negatively charged and this can interfere with the aggregation of β-LG. In fact, flower-shaped structures are only observed on mica at pH = 5, close to the isoelectric point of the protein, that is the condition that favors aggregation the most.12 Moreover, the low quality of ESEM images on mica may have in some cases prevented the detection of aggregates. Finally, we would like to point out that there is a certain degree of non reproducibility in our experiments. The formation of microscopic amorphous regions with an appropriate protein concentration depends on the complex process of drying of a droplet. Moreover, the formation of suitable aggregation seeds is an essentially random process. It is possible that small casual differences in the experimental conditions can influence this process. In particular, the growth of the structures appears to be affected by the local thickness of the protein deposit. Slow growth in thin deposit (for example around the edge of a microdroplet, Figure 5a) seems to favor flat aggregates, i.e., hexagonal crystals and flower-shaped patterns. Large rod-like structures are only observed on silicon at the center of microdroplets with a diameter g1 μm. In very large droplets, columnar

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

Conclusion A variety of protein structures have been observed for native βLG deposited on a surface. Hexagonal plates, flower-shaped patterns and elongated rods indicate that the protein self-assembles into a hexagonal columnar phase. We suggest that the anisotropic interaction with the surface leads to the formation of discotic oligomers and we hypothesize an octameric quaternary structure for these molecular building blocks. The degree of hydrophilicity of the substrate and the pH of the deposited βLG solution are both important to determine the self-organization of the protein. In particular, the most complex structures are observed on partially hydrophobic substrate (silicon) at pH below the protein isoelectric point. Further studies will be required to understand the influence of these and other physicochemical parameters relevant to aggregation, such as the protein concentration, ionic strength, temperature, and dehydration conditions. Acknowledgment. We thank Dr. Giovanni Desiderio of Licryl CNR-INFM for helpful technical assistance with the ESEM. This work was partially funded by CNR-INFM (Project MD.P01.021 “Bio-soft matter and Health Science”).

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