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Novel Amyloid Fibrillar Networks Derived from a Globular Protein: β-Lactoglobulin† Walraj S. Gosal,‡ Allan H. Clark,§ Paul D. A. Pudney,§ and Simon B. Ross-Murphy*,‡ Division of Life Sciences, King’s College London, 150 Stamford Street, London SE1 9NN, United Kingdom, and Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, United Kingdom Received January 14, 2002. In Final Form: May 20, 2002 Biology provides us with a unique set of self-assembled fibrillar networks in the form of amyloid fibrils, derived from the self-assembly of a number of peptides or misfolded proteins. These, in turn, are associated with a number of diseases such as Alzheimer’s, Creutzfeldt-Jakob disease (CJD), and type II diabetes. Recently, generating such supramolecular peptidic structures in vitro has led to a class of novel materials. In this multidistance scale, multidisciplinary study, we highlight various regimes whereby fibrils may be engineered by initiating self-assembly through the unfolding of a non-disease- associated globular protein, β-lactoglobulin (Mw ∼ 18 000, 162 residues). In particular, fibrils were generated by traditional thermal methods at pH 2, or, in a novel approach, by incubation in solvent-water mixtures such as water-2,2,2trifluoroethanol. These treatments lead to fibrils of distinct structure and morphology. Secondary structure analyses of these by Fourier transform infrared spectroscopy (FTIR) and Raman vibrational spectroscopy confirm β-sheet-mediated aggregation which is especially surprising for solvent-mediated fibril formation where an expanded helical conformation is expected. The same systems have been studied with both atomic force (AFM) and electron (EM) microscopy. The systems form gels above certain critical concentrations, which have, in turn, been characterized by rheological measurements. Again contrasts between the heatset and cold-set solvent-induced protein gels can be seen, the latter showing features reminiscent of gelatin gels.

Introduction There is renewed interest in the fibrillar gels formed from peptides and misfolded proteins. Common themes may indeed apply to self-assembled fibrillar structures, and biology itself provides us with very good examples of such structures in the form of amyloid fibrils. These mostly extracellular fibrous deposits have been implicated in a wide range of diseases known as amyloidosis, which occur due to self-assembly of a number of different proteins and peptides. At present ∼20 peptides or proteins (or fragments thereof) have been implicated in the formation of such structures in vivo, ranging in size, structure, and sequence from a small randomly coiled peptide such as β-amyloid (39-42 residues, ∼4 kDa), to the large R-helical and β-sheeted prion protein, PrP (27 kDa).1,2 In addition to such precursors, other proteins and peptides that are clearly not involved in disease can be manipulated to form amyloid fibrillar networks in vitro,3 suggesting a generic propensity for the peptide backbone to form such structures.4 Such manipulation can be achieved by de novo design of short peptide sequences, and in their simplest form, they can be constructed from † This article is part of the special issue of Langmuir devoted to the emerging field of self-assembled fibrillar networks. * To whom correspondence should be addressed: Tel +44 20 7848 4081; fax: +44 207 848 4500; e-mail simon.ross-murphy@ kcl.ac.uk. ‡ King’s College London. § Unilever Research.

(1) Sunde, M.; Blake, C. C. Q. Rev. Biophys. 1998, 31, 1. (2) Rochet, J. C.; Lansbury, P. T. Curr. Opin. Struct. Biol. 2000, 10, 60. (3) Gosal, W. S.; Ross-Murphy, S. B. Curr. Opin. Colloid Interface Sci. 2000, 5, 188. (4) Dobson, C. M. Philos. Trans. R. Soc. London, Ser. B 2001, 356, 133.

short sequences of alternating hydrophobic and charged residues.5 By increasing the hydrophobicity of the side chains, the capacity for self-assembly is also increased.5 In the case of folded proteins, self-assembly may be initiated by thermal,6 mutagen,7 solvent,8 or chemically induced unfolding of the protein.9 A less commonly employed technique is to use enzymes to degrade folded proteins in the anticipation that one or more of the fragments will self-assemble.10,11 Another strategy is the generation (either by synthesis or by recombinant expression) of peptide fragments that correspond to only part of a particular protein sequence.12,13 All of the above approaches essentially drive self-assembly, although outlining regimes under which predominantly linear (fibrillar) or amorphous aggregates occur is difficult and is, at least at the theoretical level, not well defined. In the formation of amyloid fibrils, the incentive to assemble arises from favorable solvation energies and side-chain interactions accompanying the formation of β-sheet structures.14 (5) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334. (6) Clark, A. H.; Judge, F. J.; Richards, J. B.; Stubbs, J. M.; Suggett, A. Int. J. Pept. Protein Res. 1981, 17, 380. (7) Booth, D. R.; Sunde, M.; Bellotti, V.; Robinson, C. V.; Hutchinson, W. L.; Fraser, P. E.; Hawkins, P. N.; Dobson, C. M.; Radford, S. E.; Blake, C. C. F.; Pepys, M. B. Nature 1997, 385, 787. (8) Goda, S.; Takano, K.; Yamagata, Y.; Nagata, R.; Akutsu, H.; Maki, S.; Namba, K.; Yutani, K. Protein Sci. 2000, 9, 369. (9) Yutani, K.; Takayama, G.; Goda, S.; Yamagata, Y.; Maki, S.; Namba, K.; Tsunasawa, S.; Ogasahara, K. Biochemistry 2000, 39, 2769. (10) Glenner, G. G.; Ein, D.; Eanes, E. D.; Bladen, H. A.; Terry, W.; Page, D. L. Science 1971, 174, 712. (11) Ipsen, R.; Otte, J.; Qvist, K. B. J. Dairy Res. 2001, 68, 277. (12) Guijarro, J. I.; Sunde, M.; Jones, J. A.; Campbell, I. D.; Dobson, C. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4224. (13) Wilkins, D. K.; Dobson, C. M.; Gross, M. Eur. J. Biochem. 2000, 267, 2609. (14) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; Mcleish, T. C. B.; Pitkeathly, M.; Radford, S. E. Nature 1997, 386, 259.

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However, β-sheet aggregation is also encountered in much less structured amorphous protein aggregation.15,16 The precise conditions under which certain proteins form fibrils has long been investigated.6,17-20 Here, it is well established that some net charge on the protein is essential for the formation of linear aggregates.21 The smallest sequence found to be capable of forming recognizable fibrils seems to be a pentapeptide. Scrambling of this sequence, or replacement of key residues, leads to amorphous aggregation, displaying the exact specificity of the fibril formation process.22 Structurally, fibrils present themselves as linear aggregates, typically ∼8 nm in diameter, and often several micrometers in length.23 X-ray fiber diffraction data suggests formation of an ordered central β-sheet core in which the β-sheet conformation is perpendicular to the fibril axis.24-26 The fibrils themselves are occasionally constructed from filamentous subunits or protofibrils, ∼2.5-4 nm in width, running longitudinally along the fibril axis,23 which are often arranged in a helical manner with a periodical left-handed twist around a hollow core. Here, the left-handed twist often observed for such fibrils is thought to be determined by the chirality of the amino acids,27 since constructing a fibril from exclusively D-amino acids alters this twist into a wholly right-handed one.28 Some more complex structures also have been observed such as fibril cables, ribbons or flat sheets, rings, and so forth.29,30 The precise control in assembly is, currently, only partly understood. Recently, cryoelectron microscopic work has also provided some insights into the ultrastructure of fibrils.31-33 As self-assembly proceeds, the interaction of individual amyloid fibrils, which is considered to be entirely through noncovalent interactions, may lead ultimately to the formation of a fibrous gel network.3,21 Such gels tend commonly to be relatively transparent, reflecting the narrow width of the preliminary strands, much like other

finely stranded biopolymer networks such as gelatin and pectin. Biopolymer gelation has, of course, been studied for many years, and many of the principles are well established.34-36 Both structural and mechanical (rheological) measurements have been carried out for a wide range of systems. More particularly, the many parallels between synthetic (elastomeric) gels and biopolymer gels have been clarified. Although, as mentioned above, much valuable work has been published on heat-set globular protein gelation over the preceding 50 years, this area has received renewed attention recently, by both others and ourselves.37-42 In this paper we describe amyloid fibrils formed from the bovine milk protein β-lactoglobulin (Mw ∼ 18 400, 162 residues). β-Lactoglobulin aggregation (particularly that induced by heating) has been the subject of numerous studies, although much of this has addressed so-called particulate aggregation systems (sometimes referred to as amorphous aggregation).43,44 Such a process usually occurs close to the isoelectric point (pH ∼ pI) and/or under high salt concentration. By contrast, at low pH, long linear fibrils are formed.45,46 Here we compare and contrast heatset aggregates and gels of this last type with novel fibrillar systems assembled from β-lactoglobulin under very different conditions with solvent-water mixtures such as water-2,2,2-trifluoroethanol.

(15) Oberg, K.; Chrunyk, B. A.; Wetzel, R.; Fink, A. L. Biochemistry 1994, 33, 2628. (16) Renard, D.; Lefebvre, J.; Robert, P.; Llamas, G.; Dufour, E. Int. J. Biol. Macromol. 1999, 26, 35. (17) Burke, M. J.; Rougvie, M. A. Biochemistry 1972, 11, 2435. (18) Beaven, G. H.; Gratzer, W. B.; Davies, H. G. Eur. J. Biochem. 1969, 11, 37. (19) Barbu, E.; Joly, M. Faraday Discuss. Chem. Soc. 1953, 13, 77. (20) Tombs, M. P. Faraday Discuss. Chem. Soc. 1974, 158. (21) Clark, A. H. In Functional properties of food macromolecules; Hill, S. E., Ledward, D. A., Mitchell, J. R., Eds.; Aspen Publishers: Gaithersburg, MD, 1998; p 77. (22) Tenidis, K.; Waldner, M.; Bernhagen, J.; Fischle, W.; Bergmann, M.; Weber, M.; Merkle, M. L.; Voelter, W.; Brunner, H.; Kapurniotu, A. J. Mol. Biol. 2000, 295, 1055. (23) Cohen, A. S.; Shirahama, T.; Skinner, M. In Electron microscopy of proteins; Harris, J. R., Ed.; Academic Press: New York, 1982; p 165. (24) Bonar, L.; Cohen, A. S.; Skinner, M. M. Proc. Soc. Exp. Biol. Med. 1969, 131, 1373. (25) Glenner, G. G.; Eanes, E. D.; Bladen, H. A.; Linke, R. P.; Termine, J. D. J. Histochem. Cytochem. 1974, 22, 1141. (26) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. F. J. Mol. Biol. 1997, 273, 729. (27) Aggeli, A.; Nyrkova, I. A.; Bell, M.; Harding, R.; Carrick, L.; McLeish, T. C.; Semenov, A. N.; Boden, N. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 11857. (28) Harper, J. D.; Lieber, C. M.; Lansbury, P. T., Jr. Chem. Biol. 1997, 4, 951. (29) Bauer, H. H.; Aebi, U.; Haner, M.; Hermann, R.; Muller, M.; Arvinte, T.; Merkle, H. P. J. Struct. Biol. 1995, 115, 1. (30) Hatters, D. M.; MacPhee, C. E.; Lawrence, L. J.; Sawyer, W. H.; Howlett, G. J. Biochemistry 2000, 39, 8276. (31) Gregoire, C.; Marco, S.; Thimonier, J.; Duplan, L.; Laurine, E.; Chauvin, J. P.; Michel, B.; Peyrot, V.; Verdier, J. M. EMBO J. 2001, 20, 3313. (32) Jimenez, J. L.; Guijarro, J. I.; Orlova, E.; Zurdo, J.; Dobson, C. M.; Sunde, M.; Saibil, H. R. EMBO J. 1999, 18, 815. (33) Jimenez, J. L.; Tennent, G.; Pepys, M.; Saibil, H. R. J. Mol. Biol. 2001, 311, 241.

Experimental Section Materials. Lyophilized β-lactoglobulin AB was purchased from Sigma Chemical Co. (product L-0130, lot number 20K7043) and used without further purification. Deionized water was used throughout, and all other reagents were of analytical grade and were used as received. The 50:50 water-alcohol solutions used in this study to induce fibrils were based upon ethanol and methanol (BDH, Poole, U.K.), propan-2-ol (May & Baker, Dagenham, U.K.), and 2,2,2-trifluoroethanol (Fluka Chemika, Dorset, U.K.). Fibril Formation. Lyophilized β-lactoglobulin was dissolved in deionized water (pH of solution 7.1) or in 20 mM phosphate buffer, pH 7. Where required, the pH was adjusted with the use of 1 M HCl, and monitored with a microelectrode (Radiometer, Crawley, U.K.). The protein solutions were filtered with the use of a 0.2 µm syringe filter (Whatman, U.K.). Subsequent systems were prepared by heating solutions [4-15% (w/w)] of β-lactoglobulin in water (pH 2) at 70-80 °C or in the different alcoholwater mixtures (10 mM phosphate buffer, pH 7 or pH 2) at 20 °C. Electron Microscopy. Samples [∼4% (w/w)] were diluted in deionized water, usually from 10- to 400-fold, to prevent excessive sample superimposition. For negative staining, after dilution, the sample was sprayed onto the support grid polyvinyl formal (FormVar, Agar Scientific, Stansted, U.K.) under a jet stream of nitrogen gas. The stain used in negative staining experiments (34) Clark, A. H.; Ross-Murphy, S. B. Adv. Polym. Sci. 1987, 83, 57. (35) Nijenhuis, K. T. Adv. Polym. Sci. 1997, 130, 1. (36) Clark, A. H. Curr. Opin. Colloid Interface Sci. 1996, 1, 712. (37) Lefebvre, J.; Renard, D.; SanchezGimeno, A. C. Rheol. Acta 1998, 37, 345. (38) Ikeda, S.; Foegeding, E. A.; Hagiwara, T. Langmuir 1999, 15, 8584. (39) Tobitani, A.; Ross-Murphy, S. B. Macromolecules 1997, 30, 4845. (40) Clark, A. H.; Kavanagh, G. M.; Ross-Murphy, S. B. Food Hydrocolloids 2001, 15, 383. (41) Kavanagh, G. M.; Clark, A. H.; Gosal, W. S.; Ross-Murphy, S. B. Macromolecules 2000, 33, 7029. (42) Kavanagh, G. M.; Clark, A. H.; Ross-Murphy, S. B. Langmuir 2000, 16, 9584. (43) Gimel, J. C.; Durand, D.; Nicolai, T. Macromolecules 1994, 27, 583. (44) Aymard, P.; Gimel, J. C.; Nicolai, T.; Durand, D. J. Chim. Phys. Phys.-Chim. Biol. 1996, 93, 987. (45) Aymard, P.; Nicolai, T.; Durand, D.; Clark, A. Macromolecules 1999, 32, 2542. (46) Kavanagh, G. M.; Clark, A. H.; Ross-Murphy, S. B. Int. J. Biol. Macromol. 2000, 28, 41.

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was ∼1% (w/v) methylamine tungstate. Excess stain was removed by blotting with filter paper, and the grid was then left to dry for a few minutes before imaging. For heavy metal (platinum) shadowing, mica (Agar Scientific) was used as the support grid, and the procedures were similar. Imaging was achieved by transmission electron microscopy with JEOL JEM-100CXII or JEM-1200 EX II microscopes. Atomic Force Microscopy. Fibril samples [∼4% (w/w)] were diluted, typically 1:25 to 1:200 in deionized water, sprayed onto freshly cleaved mica under a jet stream of nitrogen gas, and imaged within 24 h. Alternatively, 20 µL of diluted sample was deposited on the grid and after 2 min was washed off with 0.2 mL of deionized water and allowed to dry in air for several hours. The microscope used was a TopoMetrix Explorer (TM Microscopes, Veeco Metrology Group, Cambridge, U.K.). The microscope was mounted on an air table and covered with an acoustic hood to minimize vibrational interference. The instrument was operated with the noncontact option under ambient conditions, with the 100 µm XY 9.8 µm Z type scanner. The tips used were TopoMetrix HRF 1650 (high resonance frequency ∼320 kHz) silicon probes. The actual resonance frequency of the tip was measured once the tip was attached and the laser beam was aligned with the cantilever (typical dimensions 125 × 30 × 4 µm, respectively). Data acquisition settings were dependent on the sample under imaging, but typically topology and phase images were acquired at various relative set points (between 65% and 45%) and at scan rates 2-4 times the scan range. Normally, raw images were leveled by second-order plane leveling, followed by first-order horizontal leveling. Artifacts resulting from the leveling process were confirmed to be absent by comparison of the topology image with the phase image. Vibrational Spectroscopy. Sols and gels [2-10% (w/w)] were placed on small plastic Petri dishes, to maintain a high surfaceto-volume ratio, and dried in a fume cupboard for 4 days. The resulting glassy material was lightly ground to a fine powder and stored in vials at room temperature until analysis by Raman and/or Fourier transform infrared (FTIR) spectroscopy. Confocal Raman spectroscopy was carried out with the use of a Kaiser HoloProbe 5000 Raman spectrometer (Kaiser Optical Systems Inc., Ann Arbor, MI), coupled to an Olympus microscope by fiber optics. The Raman spectrometer employed a 785 nm laser (to help avoid natural autofluorescence) and has been described in detail elsewhere.47 Typically, a powdered sample was placed in a quartz cell, and the spectrum was collected for 250 s, with the dark (instrumental) background subtracted. Heatinduced fibrils were not examined by Raman spectroscopy, due to fluorescence from these powders. FTIR analysis was carried out with the use of a Bio-Rad FTS 6000 FTIR spectrometer (Bio-Rad, Hercules, U.K.) with a liquid N2-cooled mercury cadmium tellurium (MCT) detector. The protein spectra were collected on a diamond attenuated total reflection (ATR) system (Golden Gate accessory, Specac, Orpington, UK.). The spectra were collected at 2 cm-1 resolution with 500 scans coadded. Both Raman and FTIR spectra were examined qualitatively and are presented without deconvolution or derivative analysis. Rheological Measurements on Gels. Procedures for making such measurements are now well established.48 For example, in a “gel cure” experiment we monitor, in situ, the dependence of G′ and G′′, the so-called storage and loss moduli, at constant frequency and strain, with respect to time. Following this we then typically measure the strain and frequency dependence of the same two moduli, and of tan δ (the ratio of G′′/G′) and η*, the dynamic viscosity ()G′/ω). A small volume of solution (1.25 mL) [typical concentration 9-21% (w/w)] was pipetted onto a controlled-stress oscillatory rheometer (CSL 100, TA Instruments, Leatherhead, U.K.) at 25 °C and pseudocontrolled strain oscillatory measurements (frequency ω ) 1 rad/s, strain γ ) 1%) were made at 80 or 75 °C, using the instrument’s Peltier temperature stage. For alcoholwater mixtures, samples were loaded and maintained at 20 °C. In both cases a stainless steel cone-plate geometry (20 mm radius, (47) Everall, N.; Owen, H.; Slater, J. Appl. Spectrosc. 1995, 49, 610. (48) Kavanagh, G. M.; Ross-Murphy, S. B. Prog. Polym. Sci. 1998, 23, 533.

Gosal et al. 4° angle) was employed. The gap was set at the required level (99 µm) and any excess solution was soaked away. The expansion of the cone and plate in thermal experiments was taken into account either by allowing the cone and plate to expand at the experimental temperature and presetting the gap at 25 °C to take this into account or by using thermal expansion coefficients for the geometry and adjusting the gap at intervals ∼10 °C during the temperature ramp. No differences were found between these two methods. The time to reach the experimental temperature was monitored for each experiment, the average times to reach 75 °C being 277 ( 5 s, and to reach 80 °C, 395 ( 8 s. Several modifications were made to the CSL 100 instrument to allow cure curves to be monitored at relatively long times (∼17 h). This included the use of an extra stainless steel plate (2 mm thick), cover plate, and a sample ring holder. The former plate was used to protect the rheometer bottom plate from damage from low-pH samples. The cover plate was constructed in two parts and, when used in conjunction with a solvent-trap geometry, greatly decreased the amount of evaporation. A sample-holding ring used with solvent-water mixtures, which prevented the sample from escaping from under the cone, was removed once the gap was set. Such a series of precautions are essential when making measurements on the present systems since, without appropriate modifications, almost all commercial instruments are unsuitable for examining samples containing volatile solvents. Finally, after each (time-elapsed) cure curve experiment had been completed (typically 60 000 s), a frequency sweep (0.1-100 rad s-1, strain γ ) 1%) was performed at a series of temperatures (at 55, 45, 35, and 25 °C).

Results and Discussion Fibril Morphology. After β-lactoglobulin solutions were heated at a relatively low concentration [4-5% (w/ w)] for 18 h at 80 °C, a gel of very low modulus is formed. This gel, upon cooling and subsequent small externally applied deformation, returns to an inhomogeneous state, in which it appears that gel particles are suspended in the solution. An analysis of numerous samples of this type by electron microscopic (EM) negative staining is shown in Figure 1 (panels A and B) and reveals numerous long fibrils (∼0.1-2 µm in length). The width of these fibrils was quite narrow, the average value being typically ∼8.5 ( 1.4 nm (n, or sample size, was 31). There was no detectable regular helical twist, as is often observed for insulin, calcitonin, and Aβ fibrils. On closer inspection, some much smaller and meandering or wormlike fibrils were also evident, these being typically 100-200 µm in length but very similar in width. Similar images have recently been obtained for myoglobin.49 In addition to the above heat-set gels, solvent-induced gels can also be formed from β-lactoglobulin above a critical concentration of both protein and solvent. For example, gels were formed in 50% (v/v) TFE-water mixtures at both pH 7 and pH 2, when the protein concentration was >∼8% (w/v). Here also, the underlying morphology of the aggregates forming the gels seemed to be fibrillar in nature, as shown in Figure 1 (panels C and D). In contrast to the heat-induced fibrils, TFE-induced fibrils appeared more wormlike and granular in appearance. They were typically 150-500 nm in length, although much longer fibrils were also evident. The fibrils seemed to be quite narrowly distributed in width, which was calculated to be 7.1 ( 1.6 nm (n is 51). Homogeneous gel formation is also possible with the use of methanol, ethanol, and propan2-ol-water mixtures at pH 2 and 7. Although the fibrils (width 7.2 ( 0.8, n is 34) induced in these solvents at pH 2 seemed similar in appearance to those induced in waterTFE, pH 7, aggregates formed under similar conditions but at pH 7 seemed be composed of a mixture of finer (49) Fandrich, M.; Fletcher, M. A.; Dobson, C. M. Nature 2001, 410, 165.

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Figure 3. AFM images of β-lactoglobulin fibrils induced by incubation of a 4% (w/w) solution at 80 °C, 24 h at pH 2. Panels A and B represent unfractured sample application onto mica, whereas panels C and D represent a fractured sample. The shade is directly related to the height of the feature. Scale bar represent 500 nm.

Figure 1. Negative staining electron micrographs of β-lactoglobulin fibrils induced by (A, B) 4% (w/w) 80 °C, 24 h at pH 2; (C) 4% (w/v) 50% (w/w) TFE-water pH 7, after 3 days; (D) 3.5% (w/v) 50% (w/w) TFE-water pH 2, after 1 week; (E) 2% (w/v) 50% ethanol-water pH 2, after 5 weeks; (F) 4% (w/v) 50% (v/v) ethanol-water, pH 7 after 3 days incubation. Scale bar represents 100 nm.

Figure 2. Metal shadowing of β-lactoglobulin fibrils induced by (A) incubation of a 4% (w/w) solution at 80 °C, after 24 h at pH 2, or (B) incubation of a 4% (w/v) 50% (w/w) TFE-water pH 7, after 3 days. Scale bar represents 500 nm.

filaments (width is ∼5 nm) and bundles of these (see Figure 1, panels E and F). This marked difference is currently being investigated, although a time-dependent transformation from filament into fibril has been ruled out. Nevertheless, as is shown in Figure 2, using metal shadowing, significant morphological differences exist between heat-induced (panel A) and solvent-induced fibrils (panel B). To gain some more insight, atomic force microscopy was also employed. AFM has proved to be a useful tool in the study of fibril formation,50 since its first use with β-amyloid

fibrils.51 However, here, due to the finite shape and size of the tip (usually g10 nm), tip convolution effects lead to specimens appearing larger than they truly are.52 However, developments in carbon nanotube technology have recently been exploited to obtain higher resolution of β-amyloid aggregates.52,53 Thus, it is usual to obtain height rather than diameter information. It is then assumed that the fibril is perfectly cylindrical, although this cannot always be the case.54 The atomic force images of heat-induced fibrils are presented in Figure 3 (panels A and B). These fibrils were confirmed to be generally several micrometers long. However, the heights of the major population of long fibrils were found to be 3.6 ( 0.5 nm (n is 142), in clear disagreement with EM analysis (see later). A very small number of larger fibrils were also seen, apparently assembled from two regular fibrils and having an average height of 6.5 ( 0.6 nm. It was unclear, however, whether these fibrils were genuine or simply arose from an overlaying of two separate fibrillar components. Some very fine and very long filaments were also observed (1.4 ( 0.3 nm), and also some globular species (approximate height 1.8 ( 0.4 nm). When diluted samples are applied (sprayed) onto the mica surface with a glass capillary (diameter ∼ 0.5 mm) under a stream of nitrogen gas, the seemingly entangled networks appear to fracture, and images are replaced by fragments of the original networks, as shown in Figure 3 (panels C and D). These fragmented fibrils, as expected, appear to be similar in height to fibrils in the original (50) Ding, T. T.; Harper, J. D. Methods Enzymol. 1999, 309, 510. (51) Stine, W. B.; Snyder, S. W.; Ladror, U. S.; Wade, W. S.; Miller, M. F.; Perun, T. J.; Holzman, T. F.; Krafft, G. A. J. Protein Chem. 1996, 15, 193. (52) Wong, S. S.; Harper, J. D.; Lansbury, P. T.; Lieber, C. M. J. Am. Chem. Soc. 1998, 120, 603. (53) Harper, J. D.; Wong, S. S.; Lieber, C. M.; Lansbury, P. T., Jr. Biochemistry 1999, 38, 8972. (54) Damaschun, G.; Damaschun, H.; Fabian, H.; Gast, K.; Krober, R.; Wieske, M.; Zirwer, D. Proteins: Struct., Funct., Genet. 2000, 39, 204.

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Figure 5. Comparison of (A) heat-induced fibrils at pH 2, 80 °C, and (B) solvent-induced fibrils, 50% (v/v) TFE-water mixture. The shade is directly related to the height of the feature. Scale bar represent 100 nm.

Figure 4. AFM images of fibrils induced by incubation of a 4% (w/v) solution in 50% TFE-water mixtures at pH 7. The shade is directly related to the height of the feature. Scale bar represents 250 nm.

network (2.9 ( 0.6 nm). However, fine filaments were no longer observed, although both globular (1.6 ( 0.3 nm) and larger fibrillar (5.8 ( 1 nm) species remained. TFE-induced fibrils were also investigated by AFM, as shown in Figure 4. Here, the heights of these fibrils was found to be 2.6 ( 0.4 nm (n > 50). No suggestion of lateral aggregation of smaller species was detected, although numerous globular species were found that had heights of ∼1.1 ( 0.3 nm. Morphologically the fibrils displayed a wormlike, granular appearance, similar to images obtained with EM. Some of the higher resolution images appear to show a beaded appearance, much like the β-amyloid preliminary aggregates (or protofibrils),53 the R-synuclein protofibril,55 and fibrils formed in situ by the yeast prion protein sup35.56 The beaded appearance of these fibrils can be characterized by a periodic variance in height along the fibril length (axial periodicity). Here the peak-to-peak distance of this variance was found to be ∼34.3 ( 7.4 (n is 24), and the peak to trough height was ∼0.9 ( 0.2 nm. However, this variance was not always so obvious, and for small stretches along the fibril length, the fibril surface seemed smooth with little regular variance in height. Because of the suspected protofibrillike appearance, which for the case of β-amyloid protofibrils is prone to disaggregation upon dilution,53 we carried out dilution experiments. Here the original sample was diluted 1:200, 1:50, and 1:25. We found that although the average number of aggregates per grid size decreased with dilution, there was no apparent decrease in the average length. Although, overall, the heat-induced and solvent-induced fibrils seemed morphologically distinct, as is clearly observable in the AFM images presented in Figure 5, both types seemed largely devoid of any regular subfibrillar structure such as periodic twists. However, the fibril heights from AFM did not agree with the widths obtained from EM. There may be a number of reasons for this. The interaction of the AFM tip with the fibril can be affected (55) Conway, K. A.; Harper, J. D.; Lansbury, P. T. Nat. Med. 1998, 4, 1318. (56) Xu, S.; Bevis, B.; Arnsdorf, M. F. Biophys. J. 2001, 81, 446.

by a number of factors such as ionic concentration57 and compressibility of the biological material, especially in solution.58 Nevertheless, if the globular species observed in the AFM images are taken as individual β-lactoglobulin molecules, or even aggregates, one molecule in height, and these values are corrected on the basis of the diameter of β-lactoglobulin (∼3.6 nm), much more consistent values (7.3 and 8.5 nm, respectively) are found for the heatinduced and TFE-induced fibrils. Whether such a seemingly arbitrary correction can be applied is currently being investigated. Secondary Structural Changes upon Fibril Formation. The secondary structural changes that accompany fibril formation were followed by Raman and FTIR spectroscopy. Since the alcohols used in the present study were confirmed to interfere with many of the inherent vibrational Raman modes of β-lactoglobulin (data not shown), we limited ourselves to the study of dry powders instead. This also meant that straightforward FTIR analysis, which is usually complicated by interference from the vibrational spectrum of water,59 was also possible. Protein structure is known to alter when solvent is removed; for instance, some proteins can denature upon freeze-drying.60 However, an early comparison of Raman results for crystalline and solution β-lactoglobulin revealed identical spectra,61 and this seems to be the case for certain other proteins as well.62-64 Previous Raman work on insulin fibrils65 and bovine serum albumin gels,63 for example, has also involved a study of powders, and here a direct comparison could be made to other studies where the same systems were studied in water.66 The conclusions reached were similar. In addition, powder wide-angle X-ray diffraction, as well as fiber X-ray diffraction, are also commonly used to study protein aggregate structure, in particular the crystalline nature of the cross-β conformation in amyloid fibrils. In part, this justifies our present approach, which uses dry samples to study the same phenomena. As is shown in Figure 6, bands in the region 1620-1640 cm-1 normally assigned to β-sheet structure largely (57) Goldsbury, C.; Kistler, J.; Aebi, U.; Arvinte, T.; Cooper, G. J. S. J. Mol. Biol. 1999, 285, 33. (58) Chamberlain, A. K.; MacPhee, C. E.; Zurdo, J.; Morozova-Roche, L. A.; Hill, H. A.; Dobson, C. M.; Davis, J. J. Biophys. J. 2000, 79, 3282. (59) Susi, H.; Timasheff, S. N.; Stevens, L. J. Biol. Chem. 1967, 242, 5460. (60) Fink, A. L. Folding Des. 1998, 3, R9. (61) Frushour, B. G.; Koenig, J. L. Biopolymers 1975, 14, 649. (62) Kitagawa, T.; Azuma, T.; Hamaguchi, K. Biopolymers 1979, 18, 451. (63) Lin, V. J.; Koenig, J. L. Biopolymers 1976, 15, 203. (64) Koenig, J. L.; Frushour, B. G. Biopolymers 1972, 11, 2505. (65) Yu, N. T.; Liu, C. S.; O’Shea, D. C. J. Mol. Biol. 1972, 70, 117. (66) Clark, A. H.; Saunderson, D. H. P.; Suggett, A. Int. J. Pept. Protein Res. 1981, 17, 353.

Amyloid Fibrillar Networks Derived from β-Lactoglobulin

Figure 6. Amide I FTIR spectra (1575-1725 cm-1) of (a) native lyophilized β-lactoglobulin and powdered β-lactoglobulin pH 2 aggregates dried from solutions of (b) 4% (w/v) and (c) 10% (w/v) heated sols at 80 °C for 24 h; (d) 2% (w/v), (e) 3.5 (w/v), and (f) 7% (w/v) in 50% (v/v) methanol; (g) 2% (w/v), (h) 3.5% (w/v), and (i) 7% (w/v) in 50% (v/v) ethanol; (j) 3.5% (w/v) and (k) 7% (w/v) in 50% (v/v) propan-2-ol; and (l) 3.5% (w/v) and (m) 7% (w/v) in 50% (v/v) TFE. The 2% and 3.5% (w/v) alcoholinduced samples were incubated for 40 days, and the 7% (w/w) samples were incubated for 85 days.

dominate the amide I FTIR spectrum (1600-1700 cm-1) of dried native β-lactoglobulin solution, although very weak shoulders at 1652 and 1649 cm-1 also point to the presence of R-helix and disordered structures, respectively. Upon application of the treatments used in the microscopy work to induce formation of fibrils, the spectra show a definite shift in the amide I peak to a lower frequency for both heat- and alcohol-induced fibrils, in comparison with the native protein data. This shift is clear in each case, the greatest shift being ∼15 cm-1 (to 1617 cm-1) for 7% (w/w) aggregates formed in both 50% (v/v) methanol and

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ethanol. The amide I shift found for a 4% (w/w) heat-set solution at pH 2 is in agreement with a recent similar FTIR study in D2O, where heating a solution at a concentration of 5% (w/w), and at pD 2, shifted the amide I peak to ∼1627 cm-1.46 Similar amide I shifts have been commonly reported for other aggregated protein systems, such as fibrils,7,66-70 amorphous aggregates,16,71 and in the formation of inclusion bodies,15,72 and are clearly due to changes in the relative intensities of underlying bands. Without the use of resolution enhancement methods, however, it is difficult to specify the positions and the number of components that make up the amide I band. Although components in the region 1620-1640 cm-1 are assigned to β-sheet structures, more than one β-sheet component is common for some proteins, differences in the frequencies of these reflecting subtle differences in hydrogen bonding.73 Thus, so far a complete description of the differences of these bands is not available. Despite these complications, however, it is usually agreed that a component band at ∼1620 cm-1 (and even lower in D2O) is a hallmark of intermolecular β-sheet.66 The appearance of these low-frequency β-sheet bands occurs upon aggregation, regardless of the aggregate morphology. Thus, such changes seem as relevant to fibril formation as to less ordered amorphous aggregation, although some indirect attempts have been made to associate particular spectra with aggregate morphology.67,74 The corresponding Raman amide I spectra of native and fibrillar β-lactoglobulin are shown in Figure 7. The band assigned to the native amide I band either shifts considerably in the spectra of the fibrils from ∼1665 cm-1 toward 1673 cm-1, or a slight shoulder appears at 1673 cm-1, again indicating an increase in β-sheet structure. This can, in turn, be correlated with qualitative observations on the propensity for gelation. For example, a slight shoulder appears at 1673 cm-1 (main peak remains at ∼1664 cm-1) in the spectrum of a 50% (v/v) TFE-water pH 2 sample, which failed to gel at a concentration of 7% (w/w), while a shift resulted in the spectrum of a 50% (v/v) methanol-water pH 2 sample (to 1669 cm-1), which exhibited the greatest gelling tendency. As well as the amide I band shift noted above, band narrowing is also evident, indicating the increased intensity of the β-sheet component over the others. This is presumably a result of a decrease in helical, β-turn, and disordered regions of the protein backbone; however, to confirm the relative amounts would require resolution enhancement. The Raman amide III bands (data not shown) show substantial β-sheet as well as other structures, although changes here were less useful as diagnostic indicators. There were also very few obvious changes in the skeletal region of the spectra (data not shown). Taken together, these results indicate a near-nativelike structure within the aggregate, which in the case of the well-documented helical confor(67) Fraser, P. E.; Nguyen, J. T.; Surewicz, W. K.; Kirschner, D. A. Biophys. J. 1991, 60, 1190. (68) Caughey, B. W.; Dong, A.; Bhat, K. S.; Ernst, D.; Hayes, S. F.; Caughey, W. S. Biochemistry 1991, 30, 7672. (69) Bauer, H. H.; Muller, M.; Goette, J.; Merkle, H. P.; Fringeli, U. P. Biochemistry 1994, 33, 12276. (70) Conway, K. A.; Harper, J. D.; Lansbury, P. T., Jr. Biochemistry 2000, 39, 2552. (71) Renard, D.; Robert, P.; Garnier, C.; Dufour, E.; Lefebvre, J. J. Biotechnol. 2000, 79, 231. (72) Seshadri, S.; Khurana, R.; Fink, A. L. Methods Enzymol. 1999, 309, 559. (73) Haris, P. I.; Chapman, D. Biopolymers 1995, 37, 251. (74) Nielsen, L.; Frokjaer, S.; Carpenter, J. F.; Brange, J. J. Pharm. Sci. 2001, 90, 29.

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Figure 8. Rheological cure curves, G′ (0) and G′′ (]), for (A) heat-induced gelation, 10.5% (w/w) β-lactoglobulin, pH 2 at 80 °C, and (B) solvent-induced gelation, 10% (w/v) β-lactoglobulin in 50% TFE-water, pH 7 at 20 °C.

Figure 7. Amide I Raman spectra (1575-1725 cm-1) of (a) native lyophilized β-lactoglobulin and powdered β-lactoglobulin pH 2 aggregates dried from solutions of (b) 2% (w/v), (c) 3.5% (w/v), and (d) 7% (w/v) in 50% (v/v) methanol; (e) 2% (w/v), (f) 3.5% (w/v), and (g) 7% (w/v) in 50% (v/v) ethanol; (h) 3.5% (w/v) and (i) 7% (w/v) in 50% (v/v) propan-2-ol; and (j) 3.5% (w/v) and (k) 7% (w/v) in 50% (v/v) TFE. The 2% and 3.5% (w/v) samples were incubated for 40 days, and the 7% (w/w) samples were incubated for 85 days.

mations of β-lactoglobulin formed in alcohols75-77 would require some refolding (unless the principal aggregating species was a near-native conformation such a molten globule that has been proposed for β-lactoglobulin in alcohol solutions78). Similar observations with regard to (75) Tanford, C.; De, P. K.; Taggart, V. G. J. Am. Chem. Soc. 1960, 82, 6028. (76) Townend, R.; Kumosinski, T. F.; Timasheff, S. N. J. Biol. Chem. 1967, 242, 4538. (77) Dufour, E.; Bertrandharb, C.; Haertle, T. Biopolymers 1993, 33, 589. (78) Uversky, V. N.; Narizhneva, N. V.; Kirschstein, S. O.; Winter, S.; Lober, G. Folding Des. 1997, 2, 163.

the increase of β-sheet structure measured through Raman analysis have been noted for other systems.62,66,79-81 Aggregation of homopolypeptides can also occur in a β-sheet-mediated fashion, and the conversion of R to β-polyL-lysine, at pH 10.96 and 56 °C, is accompanied by strong Raman β-sheet bands at 1670, 1240, and 1002 cm-1.82 Rheological Experiments. Figure 8 compares and contrasts the gel cure experiments for two 10% β-lactoglobulin gels, one formed by the conventional heat-set mechanism at 80 °C and at pH 2 and the other, the novel cold-set solvent-induced gel formed, in this case, with a 50% TFE-water system at pH 7 at 20 °C. The variation in pH is not, in this case, likely to be very significant, because gel cure curves for both pH 2 and pH 7 systems are quite similar, as previously reported in some depth.42 The only differences will be in the absolute values of G′ and in the gelation times (i.e., the times required for G′ to rise rapidly from its solution value) attained, but these will also tend to depend on the precise thermal history and the concentration of protein. In this respect the (upper) trace observed for the heatset gelation system is quite typical. At the start of the experiment G′ and G′′ are both very low, but interestingly G′ > G′′. This is not a typical observation for a gelling system, since for a solution we would normally expect the converse behavior.48 However, it has been previously been reported by ourselves and others39 and attributed to colloidlike structuring in the pregel solution. This aspect (79) Nonaka, M.; Lichan, E.; Nakai, S. J. Agric. Food Chem. 1993, 41, 1176. (80) Lichan, E.; Nakai, S. J. Agric. Food Chem. 1991, 39, 1238. (81) Painter, P. C.; Koenig, J. L. Biopolymers 1976, 15, 2155. (82) Yu, T. J.; Lippert, J. L.; Peticolas, W. L. Biopolymers 1973, 12, 2161.

Amyloid Fibrillar Networks Derived from β-Lactoglobulin

Figure 9. Rheological frequency sweeps, G′ (0), G′′ (O), and η* (4), for (A) heat-induced gelation, 10% (w/w) β-lactoglobulin, pH 2 at 75 °C, and (B) solvent-induced gelation, 10% (w/v) β-lactoglobulin in 50% TFE-water, pH 7 at 20 °C.

has recently been addressed in more detail by Ikeda and Nishinari,83 who have shown that the effect is genuine but is not due to classical (DLVO) intercolloidal forces, and occurs only if “a nonelectrostatic large repulsive interaction exists”. Interestingly, for the solvent-induced system, this effect is not observed, and although values are very low, it does appear that the more usual G′′ > G′ pregel behavior pertains. Whether this is simply due to the lower dielectric constant of the 50-50 mixed solvent, or to other factors, remains to be determined. On subsequent heating both G′ and G′′ increased very substantially at the gel point, a conventional crossover being found for the solvent-based system but not for the heat-set gel, as already discussed. Both systems then reach an apparent plateau in G′ after ∼104 s. (The slight apparent decrease in G′ seen after very long times is almost certainly a gel shrinkage artifact, as discussed elsewhere.42) Perhaps the most interesting difference between the two systems is, however, the clear maximum in G′′ seen in the solvent-induced gel. Such behavior is very commonly seen for synthetic gels and sometimes even for gelatin gels84 but never, in our experience, for conventional heat-set protein gels. According to Bibbo and Valles,85 it is an effect that can be associated with the relaxation of “dangling chain ends”. Qualitatively, we have asserted that this, in turn, reflects a greater degree of chain flexibility in the underlying gel network.34 (83) Ikeda, S.; Nishinari, K. Food Hydrocolloids 2001, 15, 401. (84) Ross-Murphy, S. B. Rheol. Acta 1991, 30, 401. (85) Bibbo, M. A.; Valles, E. M. Macromolecules 1984, 17, 360.

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Figure 9 shows the corresponding frequency spectrum of G′ and G′′ for the same gels after completion of the gel cure experiment. Both systems show typical gel spectra, with G′ showing only very little frequency dependence, so we can assert that the storage modulus will have a finite value even at very long times (low frequencies); i.e., it shows solidlike properties. This is of real significance, because many of the systems commonly referred to in the literature as “gels” are simply very high viscosity fluids or entangled solutions. Furthermore, without such data, it is not possible to distinguish gels from other classes of materials. To those involved in research in polymer gels and networks this is self-evident, but it may not be so for workers more involved in structural protein fibril studies. Finally we note again the major contrast seen in the behavior of G′′ for the heat-set and the solvent-induced gels. For the former, G′′ is also essentially frequencyindependent (the slight notchiness seen here is most likely an instrument resonance artifact). By contrast, the spectrum for the solvent gel shows a slight gelatinlike minimum in the middle range of oscillatory frequencies. Furthermore, the ratio of G′/G′′ is significantly smaller at most frequencies. Although such qualitative comparisons are somewhat dangerous, this again suggests a more tenuous structure with more pronounced internetwork chain flexibility. In this respect, at least, the rheological data are quite consistent with the evidence from microscopy. More quantitative analysis of the concentration dependences of the gelation time and modulus of the gels is underway, although this requires a more detailed approach, as outlined in other recent papers.40,42 Conclusion Results for the novel solvent-induced gels formed from β-lactoglobulin are contrasted with those for the more conventional heat-set fibril gels formed at low pH. The former show features more usually attributed to molecular networks and may be useful model systems for future studies of fibrillar gels, either the amyloid type associated with disease or novel materials constructed from peptide self-assembly.3 It is also interesting to determine, as we suspect, whether such solvent-induced protein gels can be formed from other globular proteins. Certainly heatset gelation as a route to forming fibrillar networks is a well-established, even historic route. The new systems show both similarities and differences with the heat-set systems, which may prove a fruitful line for further investigations, for example, of structure-property relationships. Acknowledgment. We thank John Pacey (King’s College) and Anthony Weaver (Unilever Research) for assistance with electron microscopy experiments; Dr. XueFeng Yuan, Dr. Yung Jong Lee, and Dr. Paul Royall (King’s College London) for helpful advice and access to the atomic force microscope; Dr. Alex. F. Drake (King’s College) for advice on FTIR experiments; Dale G. Cunningham (Unilever Research) for experimental FTIR/Raman data; and Barry Taylor (also King’s College) for careful design and construction of accessories for the Carri-med 100 instrument. W.S.G. thanks the (U.K.) Biotechnology and Biological Sciences Research Council (BBSRC) and Unilever Research for the award of a CASE studentship. LA025531A