Experimental Evidence for Multiple Assembled States of Sc3 from

Biomacromolecules 2003, 4, 956-967

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Experimental Evidence for Multiple Assembled States of Sc3 from Schizophyllum commune Paul A. Stroud,† J. Shawn Goodwin,‡ Peter Butko,‡ Gordon C. Cannon,‡ and Charles L. McCormick*,†,‡ Department of Polymer Science and Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi 39406-0076 Received February 11, 2003; Revised Manuscript Received April 25, 2003

The hydrophobin Sc3 from the fungus Schizophyllum commune assembles from the aqueous phase into ordered structures with substantially different characteristics depending upon experimental conditions. Under the first condition, a vortexing procedure widely reported in the literature, interfacial assembly yields highly ordered, stacked β-sheets. We have also observed a previously unreported assembly of Sc3 under a second condition, which occurs in a time-dependent manner from quiescent solution. The resulting types of assembled states have been compared utilizing fluorescence techniques, sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, density gradient centrifugation, and phase contrast and atomic force microscopy. A model based on this study and previous literature is proposed that suggests three distinct states of Sc3: (1) soluble Sc3 consisting of unimers or multimers in micelle-like association, (2) interfacially assembled I-Sc3 with highly ordered, stacked β-sheets, presumably formed in a templated manner at the air/water interface of microscopic bubbles generated by vortexing, and (3) solution-assembled S-Sc3, a less-ordered structure formed in a time-dependent manner in the absence of an interface. Introduction Hydrophobins are a group of amphipathic fungal proteins possessing remarkable structural characteristics that allow for unique self-assembling behavior. These characteristics have spawned a flurry of research activity including that focused on pharmaceutical and other medical applications.1-5 Of primary interest is the ability of hydrophobins dissolved in aqueous media to readily organize into insoluble amphipathic films at hydrophobic/hydrophilic interfaces. Hydrophobins are low molecular weight (8-10 kDa) polypeptides that contain eight conserved cysteines in their sequence. The conserved cysteines are intramolecularly cross-linked and are considered important for maintaining protein solubility and avoiding premature self-association.1 Hydrophobins can be characterized as either class I or class II on the basis of their solubility characteristics and hydropathy patterns,2-4 yet both classes show a similar propensity to self-assemble into approximately 10 nm thick amphipathic insoluble membranes.5 Class I films are not disrupted when treated with surfactants, solvents, and denaturing agents (e.g., urea).4,6 Only trifluoroacetic acid (TFA) has been found to dissociate these supramolecular structures.6 By contrast, class II films are easily disrupted with surfactants or solvent mixtures such as 60% ethanol/ water.7 Though many class I and class II hydrophobins have been identified and isolated, the most well-studied hydro* To whom correspondence should be addressed. E-mail: Charles. [email protected]. † Department of Polymer Science. ‡ Department of Chemistry and Biochemistry.

phobin to date is Sc3, a class I hydrophobin from the fungus Schizophyllum commune.4-6,8-11 When air/water interfaces are created in hydrophobin solutions (i.e., by bubbling gas through the solution), proteincoated air bubbles result that can be visualized by light microscopy.6,7 Hydrophobins also stabilize oil droplets in solution.11,12 For example, Sc3 and oil mixtures extruded through membranes of fixed pore sizes result in uniform protein-coated vesicles.12 In addition to the stabilization of bubbles and oil droplets, hydrophobins can modify both hydrophobic and hydrophilic surfaces as applied from aqueous solution. Preferential surface orientation arises from the amphipathic nature of Sc3.10-14 Amino acid sequences of hydrophobins contain distinct hydrophilic and hydrophobic regions, and it has been proposed that hydrophobins may resemble a polymeric surfactant with high surface activity.10,11,15-17 The hydropathy plot of Sc3 indicates that approximately the first third of the amino acid sequence from the N terminus is relatively hydrophilic compared to the remaining portion.2,3 In addition, Sc3 contains approximately 20 mannose residues, thought to be O-linked through threonines in the N-terminal third of the protein, further increasing the hydrophilicity of that region of the polypeptide chain.10 The Sc3 protein in its unimeric or “unassociated” form is comprised of a high proportion of β-sheet structure (41%), as determined by circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy.4,5,10 When Sc3 is induced to self-organize at the air/water or Teflon/water interface, a small, transient increase in R-helical secondary structure is observed, followed by an increase in β-sheet structure. The

10.1021/bm034045e CCC: $25.00 © 2003 American Chemical Society Published on Web 06/05/2003

Evidence for Multiple Assembled States of Sc3

Sc3 self-assembly was proposed to be a multistep process in which the R-helical state is intermediate between the unassociated protein and the β-sheet state.4,5,10 One distinguishing feature of class I hydrophobins is the characteristic 5-12 nm rodlet structure observed on the hydrophobic side of an amphipathic protein film.6,11,18 These rodlets are presumed to be insoluble and have not been shown to exist in solution. Hydrophobin rodlets have been proposed by some authors to be similar to amyloid fibrils of misfolded proteins observed in prion or amyloid diseases.1,4,5,18-21 These structures consist of stacked β-sheets arranged perpendicular to the fibril axis.22,23 Interactions between the stacked β-sheets can occur along the entire length of the fibril, creating structures that are extremely stable and difficult to dissociate.20 The fluorescent probe thioflavin T (ThT), which specifically associates with β-sheet stacks such as those seen in amyloid fibrils,24 binds to the self-assembled Sc3.21 Likewise, the amyloid-specific dye Congo Red interacts with self-assembled Sc321 and displays a gold-green birefringence pattern when mixed with assembled structures of the EAS hydrophobin.18 The mechanism of hydrophobin assembly, including orientation and deposition at an interface, has not been fully elucidated. In addition, it is unclear whether unassociated protein molecules interact in the absence of a hydrophobic interface. To clarify some of these issues, experiments were conducted to compare self-assembled structures formed upon vigorous agitation (introducing air bubbles by vortexing) with the largely unstudied structures formed under quiescent conditions. Techniques including atomic force microscopy (AFM), light microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), gradient centrifugation, and fluorescence spectroscopy have thus been utilized to distinguish two pathways of Sc3 assembly. One pathway requires orientation at the air/water interface resulting in the formation of highly ordered structures, whereas the other pathway is a slow, noninterfacially driven process. This study supports the considerable literature reports of other groups and allows extension of a previous model17 by postulating the existence of a soluble unimeric state, a highly ordered stacked β-sheet state from interfacial assembly, and a lessordered state resulting from time-dependent association in water. Experimental Section Materials. Trifluoroacetic acid (TFA) was purchased from VWR Scientific products (West Chester, PA). Forty percent aqueous acrylamide/bisacrylamide (29/1) solutions, ammonium persulfate, nitrocellulose, and glycine were purchased from BioRad (Hercules, CA). Mica sheets (1 cm × 4 cm) were purchased from Ted Pella (Redding, CA). All other reagents used were of the highest purity available. Protein Purification and Preparation. The fungus Schizophyllum commune was grown in a 1 L flask in minimal medium as described by Dons et al.25 Protein was purified by a method adapted from a published protocol utilizing hydrophobic interaction chromatography.17 Protein resulting from column purification was agitated thoroughly in 1.5 mL

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microcentrifuge tubes (1 mL of protein solution) with a benchtop vortexer for 20 min. The resulting turbid Sc3 solution was centrifuged with a tabletop microfuge at 14,000 rpm for 20 min. The supernatant was discarded, and the pellet from each tube was dissolved in 0.5 mL of TFA, resulting in a clear solution. The protein samples dissolved in TFA were combined in a 25 mL glass scintillation vial, dried with a stream of nitrogen, and lyophilized for at least 12 h to complete dryness. SDS-PAGE Analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli26 with the Bio-Rad Mini-Protean III apparatus. All gels were stained with a BioRad Silver Stain Plus kit following the manufacturer’s instructions. Aliquots of Sc3 were added to 4× Laemmli loading buffer, heated at 95 °C for 5 min, and allowed to stand at room temperature until gels were loaded. Densitometry. Spot densitometry was performed with an AlphaImager 2000 Documentation and Analysis System from Alpha Innotech Corporation (San Leandro, CA). All densitometry values were normalized to the time-zero fraction or to the load (diluted by a factor of 5) of the sucrose density gradients on the polyacrylamide gels and immunoblots. Protein Assay. Protein concentrations were determined utilizing the bicinchoninic acid (BCA) assay from Pierce (Rockford, IL). Bovine serum albumin from Sigma (St. Louis, MO) was used as the standard. Immunoblot Analysis. Protein samples were deposited onto nitrocellulose membranes using the Bio-Rad Bio-Dot SF apparatus. The nitrocellulose membrane and filter paper were presoaked in phosphate-buffered saline (PBS) for 10 min at room temperature. Each sample reservoir was then prewashed with 250 µL of PBS under vacuum. Protein samples were loaded into the reservoirs, applied onto the membrane via vacuum, then washed with 250 µL of PBS, and subsequently dried. To immunologically detect Sc3 that was bound, the membrane was washed with PBS containing 0.1% Triton-X-100 and 5% w/v evaporated milk for 45 min. The membrane was then treated with 1:1000 dilution of a rabbit anti-Sc3 serum in PBS/Triton-X-100 for 1 h. The membrane was washed with PBS/0.1% Triton-X-100, PBS/ 0.1% Triton-X-100/5% milk, and PBS/0.1% Triton-X-100 for 15 min each before being incubated with a 1:10,000 dilution of a goat anti-rabbit IgG conjugated with alkaline phosphatase (Sigma) in PBS/0.1% Triton-X-100 for 1 h, and washed as above. The immunoblot was developed using a one-step NBT/BCIP solution (Pierce). Phase-Contrast Light Microscopy. Aliquots (20 µl) of Sc3 solutions were spotted onto clear glass slides and overlaid with a cover slip. Images were taken with an Olympus BX60 microscope (Melville, NY) in phase-contrast mode and recorded with an Optronics DEI-470 camera (Goleta, CA) utilizing Scion Image capture software (Frederick, MD). Steady-State Fluorescence Spectroscopy. Fluorescence spectra were recorded at 25 °C with an Edinburgh TGeometry fluorimeter (Edinburgh, Scotland). Slit widths were set at 10 nm in both the excitation and emission monochromators. All spectra were corrected for light scattering by

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Figure 1. Assembly of Sc3 (A) monitored with SDS-PAGE by the decrease in the 24 kDa band (U-Sc3): lane 1, Bio-Rad broad range molecular weight standards; lane 2, aliquot of the I-Sc3 sample; lane 3, aliquot of U-Sc3 sample; lanes 4-8, aliquots taken from the undisturbed S-Sc3 samples over time (24-120 h). Panel B shows immunoblot analysis of Sc3 assembly: lane 1, aliquot of the I-Sc3 sample; lane 2, aliquot of the U-Sc3 sample; lanes 3-7, aliquots of undisturbed S-Sc3 over time (24-120 h). Panel C shows total protein concentration of aliquots taken from the U-Sc3 (0 h) and the undisturbed S-Sc3 (24-120 h) samples over time. Error bars represent the standard deviation of three measurements. Panel D shows quantitative densitometry of the 24 kDa Sc3 bands after SDS-PAGE and Sc3 bands after immunoblot analysis (panels A and B, respectively) resulting from the I-Sc3 samples from panel A and panel B. Lane 2 in panel A has a value of 0.03, whereas lane 1 in panel B has a value of 0.90.

subtracting a spectrum of the sample without probe. For studies using 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt (bis-ANS, Molecular Probes, Eugene, OR), the samples were excited at 395 nm and spectra were recorded from 410 to 650 nm. For analyses using the probe ThT (Sigma, St. Louis, MO), samples were excited at 435 nm and emission was monitored from 450 to 600 nm. Congo Red Absorption. Congo Red absorption measurements in the presence of Sc3 were performed as previously published.21 Atomic Force Microscopy. Freshly cleaved mica sheets (1 cm × 4 cm) were sequentially rinsed three times each with ethanol and water and then allowed to dry in covered containers to prevent deposition of air-borne contamination. A solution of purified Sc3 in duplicates (200 µL) was added at each time point onto the mica and placed on a constant temperature hotplate (55 °C) to ensure that the sample was dry within 2-3 h. Samples were imaged in air at several locations on the coated surface with a Dimension 3000 scanning probe microscope (Digital Instruments, Santa Barbara, CA) with a 125 µm silicon cantilever in tapping mode. Density Gradient Centrifugation. Linear 10%-60% sucrose gradients (13.5 mL), buffered with 20 mM sodium phosphate, pH 7.5, were prepared using an Instrument Specialties Company model 570 gradient maker (Lincoln, NB). Samples (0.75 mL) were loaded on top of the gradients and centrifuged in a Beckman L8-80 ultracentrifuge that was equipped with an SW40Ti rotor (Beckman, Fullerton, CA) for 40 min at 114 000 × g. Ten gradient fractions were collected and dialyzed extensively against water. Aliquots

from each fraction were analyzed by SDS-PAGE and immunoblotting before dialysis. Protein concentration was determined by the BCA assay. Results The objectives of this research were to examine the selfassembly of the class I hydrophobin Sc3 under two specified conditions, compare the resulting higher-order structures, and refine a previously published mechanistic model of selfassembly from aqueous media. In this section, we describe assembly under condition 1, involving organization at an air/ water interface created by vigorous vortexing, and condition 2, allowing an undisturbed, time-dependent self-association from aqueous solution. Experiments utilizing SDS-PAGE, light microscopy, steady-state measurements with selected fluorescence probes, atomic force microscopy, and density gradient centrifugation were conducted to determine the nature, size, stability, hydrophobicity, and time dependence of formation of the higher-ordered structures. Two Assembled States of Sc3. Hydrophobins, especially Sc3, are prone to interfacial self-assembly in response to even the slightest agitation of the polypeptide solution. Thus, prior to each experiment, Sc3 was treated with 100% TFA and then lyophilized, a process reported to disassemble any Sc3 complexes.6 The resulting lyophilized hydrophobin was then dissolved in the appropriate buffer to obtain a clear solution of soluble protein, which we shall refer to as unimeric-Sc3 (U-Sc3). U-Sc3 appears as a single band in SDS-PAGE gels at the apparent molecular weight of 24 kDa (Figure 1A, lane 3).


Figure 2. Protein concentrations of centrifuged aliquots taken from U-Sc3, S-Sc3 (24-120 h), and I-Sc3 samples: (9) supernatants of centrifuged S-Sc3 samples; (b) pellets of centrifuged S-Sc3 samples; (O) supernatant of I-Sc3 sample; (0) pellet of I-Sc3 sample. Error bars represent the standard error of three measurements.

SDS-PAGE (Figure 1A) was also utilized to monitor the association of U-Sc3 (18 µg/mL) into higher-order SDSinsoluble complexes from an undisturbed sample over a period of 120 h. Aliquots were withdrawn at regular intervals and studied by SDS-PAGE. The results clearly indicated that the intensity of the 24 kDa protein band (U-Sc3) decreased with time (Figure 1A, lanes 3-8). The portion of the aliquot that did not enter the gel, the SDS-insoluble Sc3, will be referred to as S-Sc3 for solution-assembled Sc3. The U-Sc3 solution (18 µg/mL) that had been vortexed showed no evidence of a soluble fraction (Figure 1A, lane 2), which could not enter the gel, a result consistent with that reported previously by Wo¨sten et al.6 As will be shown later, molecular architecture of this vortexing-induced material differs from that of S-Sc3 and will be referred to as I-Sc3 (interfacially assembled Sc3). Aliquots from the same samples analyzed by SDS-PAGE were applied directly onto a nitrocellulose membrane by vacuum, and the immunoblot was developed after incubation with rabbit anti-Sc3 serum (see Experimental Section) (Figure 1B). Because both soluble and insoluble phases were sampled, the immunoblot (Figure 1B) shows that approximately equal amounts of total protein were present in all of the aliquots of the U-Sc3, S-Sc3 (0-120 h), and I-Sc3 samples. Likewise, determination of protein concentration utilizing the BCA assay indicated no detectable change of total protein in each aliquot (Figure 1C). The bands from the gel (Figure 1A) and immunoblot (Figure 1B) were quantified by spot densitometry, and the results were plotted (Figure 1D). Vortexing (condition 1) converted nearly all Sc3 into the assembled, insoluble I-Sc3 form that was unable to enter gel. By contrast, under condition 2, progressive, slow assembly into S-Sc3 was evidenced by lower amounts of unimeric Sc3 entering the gel from aliquots taken at longer times. Analysis of Sc3 Complexes by Centrifugation. Aliquots of the U-Sc3, S-Sc3 (24-120 h), and I-Sc3 samples were subjected to centrifugation. The protein concentrations in the resulting supernatants and pellets were measured by the BCA assay (Figure 2). During S-Sc3 assembly (condition 2), the concentrations of Sc3 in the supernatants decreased from the initial value of 30.8 µg/mL (U-Sc3 sample, 0 h) to 17 µg/ mL (S-Sc3 sample, 120 h). Those S-Sc3 complexes large enough to sediment were measured in the pellets at concen-


trations as high as 5.5 µg/mL of Sc3 in the 120 h S-Sc3 sample. By contrast, the I-Sc3 sample (condition 1) after centrifugation had only 5 µg/mL in the supernatant and 23 µg/mL in the pellet (Figure 2). When converted to percentages in each phase, the I-Sc3 sample contained 82% of the Sc3 in the pellet and 18% in the supernatant, whereas the S-Sc3 (120 h) sample contained 22% of Sc3 in the pellet and 78% in the supernatant. Density Gradient Centrifugation. Density gradient centrifugation was utilized to qualitatively study the size distributions of U-Sc3, S-Sc3, and I-Sc3 (120 h) in solution. Aliquots of each sample were loaded onto 10%-60% sucrose gradients and centrifuged, and the gradients were fractionated. The quantities of Sc3 in each fraction were then determined utilizing the BCA assay. Samples from each fraction were subsequently analyzed for soluble Sc3 (U-Sc3) by SDS-PAGE and for complexed Sc3 (I-Sc3 or S-Sc3) utilizing immunoblot analysis. Figure 3A indicates that protein was predominantly located in the upper fractions of the gradient in the U-Sc3 sample. The first three fractions contained 25, 16, and 8.3 µg/mL, respectively, accounting for 48% of the total protein (Figure 3A). In addition, a 24 kDa band was observed in fractions one through three after SDS-PAGE (gel not shown), but no bands were observed in the later fractions (fractions 4-10 and pellet). Figure 3B illustrates the behavior of the 120 h S-Sc3 sample (condition 2). A difference in distribution within the gradient is readily observed by comparison with Figure 3A. For the S-Sc3 (120 h) sample, only 30% of the total protein was present in the first three fractions at concentrations of 11.1, 10.3, and 3.2 µg/mL, respectively. After SDS-PAGE, 24 kDa bands were observed only in the first two fractions of the gradient (not shown). Protein in the I-Sc3 sample was distributed throughout the gradient (Figure 3C), 12% having sedimented to the bottom of the centrifugation tube (compared with 4% and 6% in panels A and B of Figure 3, respectively). Sc3 in the I-Sc3 sample was present entirely in SDS-insoluble complexes because 24 kDa bands were not observed after SDS-PAGE in any gradient fraction. Light Microscopy. Light microscopy was used to directly visualize S-Sc3 (120 h) and I-Sc3. The undisturbed sample of S-Sc3 at 120 h contained large (20-200 µm), irregularly shaped complexes (Figure 4A). The vortexed sample (I-Sc3), which was extremely turbid, contained largely spherical structures, presumably protein-coated air bubbles, of various sizes up to 25 µm (Figure 4B), consistent with reports by Wo¨sten et al.6 Atomic Force Microscopy. Atomic force microscopy (AFM) was used to view Sc3 deposited on freshly cleaved mica. After the U-Sc3 sample was dryed onto the mica, the characteristic 10 nm rodlet pattern was observed (Figure 5A), which is consistent with published results.10 Aliquots taken from the S-Sc3 sample (containing U-Sc3 and S-Sc3 complexes) were dried onto mica over a period of 120 h and imaged. After 24 h, complexes that were globular in appearance were present on the mica surface in addition to the rodlets (Figure 5B). After 120 h, the S-Sc3-coated mica surface was covered with a higher number of globular

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Figure 3. Density gradient centrifugation of aliquots taken from U-Sc3, S-Sc3 (120 h), and I-Sc3 samples. Immunoblot analysis, quantitative densitometry, and protein concentrations of 10%-60% sucrose gradient fractions of the (A) U-Sc3 sample, (B) S-Sc3 (120 h) sampele, and (C) I-Sc3 sample are shown. Error bars represent the standard error of three measurements of Sc3 concentrations in each gradient fraction. The gradient fractions for each sample are indicated by 1-10 and pellet (P). Bands from each immunoblot were normalized to the value of the load (L) for each gradient.

complexes, some quite large in size (Figure 5C). Rodlet structures were also present on the mica surface but in lower quantities than in the 0 and 24 h S-Sc3 samples (Figure 5C). By contrast, aliquots of I-Sc3 that were dried onto mica did not produce rodlets and had a rougher appearance (micrographs not shown). Bis-ANS Fluorescence in the Presence of Sc3. The fluorescence dye bis-ANS was utilized to probe the surface hydrophobicity of the unimeric and assembled forms of Sc3 in solution. The fluorescence intensity of bis-ANS increased slightly in the presence of U-Sc3 as compared to that in buffer alone (Figure 6A). In the presence of vortexed Sc3 (I-Sc3), however, the fluorescence intensity of the probe increased 5-fold over that shown with the U-Sc3 sample (Figure 6A). In addition, the λmax shifted from 535 to 485 nm. By contrast, when bis-ANS was added to aliquots taken from the S-Sc3 sample, the fluorescence intensity did not increase but fell below that seen with the U-Sc3 sample and by 120 h approached that of the buffer alone (Figure 6A).

Figure 6B clearly shows this decrease in fluorescence intensity with time. Thioflavin T Fluorescence in the Presence of Sc3. The fluorescence probe ThT was utilized to examine ordering of Sc3 into stacked β-sheet structures under conditions 1 and 2. Fluorescence intensity was monitored at 485 nm27 in the presence of U-Sc3, S-Sc3, and I-Sc3. The fluorescence intensity of ThT in the presence of I-Sc3 was 50-fold higher than that in the presence of U-Sc3 (Figure 7A). By contrast, the fluorescence intensity of ThT in S-Sc3 (24-120 h) aliquots increased only to a small extent as compared to the effect seen with the U-Sc3 sample (Figure 7B,7C). To determine whether S-Sc3 assemblies could be converted to I-Sc3 structures, the 168 h S-Sc3 sample was vortexed (I-Sc3 (168 h)) and mixed with ThT, and the fluorescence was measured and compared to the Sc3 sample of the same concentration that was vortexed at the 0 h time point (I-Sc3 (0 h)) (Figure 7D). The fluorescence intensity of ThT in the presence of the S-Sc3 (168 h) sample was


Figure 4. Light micrographs of (A) an aliquot taken from the S-Sc3 sample at 120 h and (B) an aliquot taken from the I-Sc3 sample. Bar ) 10 µm.

comparable to that seen with the U-Sc3 sample but increased in the two vortexed samples (I-Sc3 (0 h) and I-Sc3 (168 h)) (Figure 7D). The Effect of Preassembled Sc3 on S-Sc3 Assembly. The presence of stacked β-sheets in the interfacially assembled Sc3 (I-Sc3) and the lack of such structures in the solution-assembled Sc3 (S-Sc3) point to different assembly pathways under conditions 1 and 2, respectively. Hydrophobin self-assembly has been compared to amyloidogenesis.1,18,21 The characteristic feature of amyloid fibrillogenesis is that the addition of preassembled amyloid fibrils (seed or nucleus) to unassembled protein increases the rate of assembly in vitro.20 Also, an increase in amyloidogenic protein concentration accelerates the nucleation rate and the rate of the subsequent assembly into fibrils.20 Therefore, the effects of addition of preassembled Sc3 and the concentration dependence of the S-Sc3 assembly rate were studied. The effect of “seed” on S-Sc3 assembly was assessed by adding three forms of preassembled Sc3 to solutions of U-Sc3. The first type of seed was Sc3 that had sedimented from an S-Sc3 (120 h) sample. The second seed was an aliquot taken from an I-Sc3 solution. The third seed was U-Sc3 that had been dried onto the interior of the glass vial in which the seeding experiment was performed. Two concentrations of each type of seed were added to U-Sc3 samples. The amounts of the remaining U-Sc3 were measured after taking aliquots over a 120 h period and performing SDS-PAGE. Panels A and B of Figure 8 are silverstained polyacrylamide gels of U-Sc3 solution to which 2.5 and 5.0 µg, respectively, of the I-Sc3 seed were initially added. Quantification by densitometry of the 24 kDa bands was performed, and the results were plotted (Figure 8CE). The addition of “seed” in all three cases inhibited the


rates of assembly (more U-Sc3 remained in solution) relative to those observed for the unseeded S-Sc3 control samples. Figure 8C-E demonstrates that higher seed concentration leads to greater inhibition. Concentration Dependence of Sc3 Assembly. The rate of U-Sc3 assembling into S-Sc3 structures (condition 2) was studied as a function of the initial concentration of U-Sc3. Samples containing four different concentrations of U-Sc3 were prepared, and aliquots taken over 120 h were analyzed by SDS-PAGE. To compare the rate of assembly, the same total protein concentration from each aliquot was loaded onto the gels. The resulting bands after staining were quantified by densitometry, and the values were compared (Figure 9A). Increasing the initial concentration of U-Sc3 in solution resulted in a decrease in the rate of conversion of U-Sc3 into S-Sc3 complexes (Figure 9). The percentage of U-Sc3 assembly into I-Sc3 structures was studied by SDS-PAGE analysis as a function of the initial U-Sc3 concentration ranging from 2.5 to 27.5 µg/mL. The samples were subjected to vortexing, and aliquots of both were analyzed by SDS-PAGE. The intensities of the 24 kDa bands were determined by densitometry, and those of vortexed I-Sc3 bands were normalized to the 100% U-Sc3 bands at each concentration. Figure 9B demonstrates that the relative amount of I-Sc3 increased with increasing Sc3 concentration, whereas the amount of S-Sc3 structures decreased. Discussion The results of the studies reported here and in previous reports are consistent with a model for Sc3 assembly shown in Figure 10. Sc3 exists as unimers or loosely associated aggregates in aqueous solution (U-Sc3). The protein’s high surface activity is due to the amphipathic character of its amino acid sequence. This, coupled with vortexing, results in rapid transport to the air/water interface. At this interface, Sc3 apparently orients and assembles into stable (SDSinsoluble) amphipathic membranes (I-Sc3). In the absence of an interface, Sc3 slowly assembles over time into large, irregularly shaped, also SDS-insoluble structures (S-Sc3), which are distinct from the I-Sc3 complexes. Unimeric Sc3. Treatment of Sc3 with TFA disrupts interactions between the polypeptide chains, thus allowing for the solvation of unimeric Sc3 protein in aqueous media.5,6,28 We refer to the Sc3 hydrophobin treated in this manner as unimeric-Sc3 (U-Sc3) for simplicity, indicating that all Sc3 molecules are unassociated in solution. In reality, U-Sc3 may be a mixture of free, unassembled Sc3 and loosely associated, possibly micelle-like oligomers or multimers that are SDS-soluble (Figure 10). We have previously shown that the polysaccharide schizophyllan that is coproduced in aqueous media by S. commune prevents aggregation under nonvortexing conditions. Hydrophobins, in the absence of schizophyllan or stabilizing molecules such as surfactants or TFA, possess a sufficient number of hydrophobic amino acids that may direct self-association in solution at extremely low concentrations. While the number of associating molecules is certainly low, Sc3 is one of the most surface-active

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Figure 5. Atomic force microscopy of Sc3 dried onto mica. Images are height and amplitude (right and left, respectively) for each sample: (A) aliquot taken from the U-Sc3 sample (setpoint ) 0.7918 V; scan rate ) 1.001 Hz); (B) aliquot taken from the S-Sc3 sample (24 h) (setpoint ) 0.7414 V; scan rate ) 1.001 Hz); (C) aliquot taken from the S-Sc3 sample (120 h) (setpoint ) 0.7068 V; scan rate ) 0.7535 Hz.

proteins known.15 Multiple Sc3 chains also appear to associate into supramolecular aggregates, especially at high concentrations.17 Evidence supporting an equilibrium between unassociated Sc3 and larger aggregates was provided by Martin et al.17 utilizing dynamic light scattering. Although no unimers were detected in that work because of limitations of the laser used in the scattering studies, complexes ranging from approximately 50 to 100 nm were demonstrated in solution at low concentrations of the protein (1 µg/mL). At higher concentrations (4-10 µg/mL), an additional population of aggregates in the micrometer range was observed. The size of the loosely associated structures in solution was shown to be dependent on the protein concentration; surface tensiometry experiments showed a critical aggregation

concentration of approximately 1 µg/mL. Interactions between Sc3 molecules have been indicated by energy transfer in solution between N-terminally labeled Sc3 in the U-Sc3 or “soluble” state.28 In addition, small-angle X-ray diffraction (SAXD) and size exclusion chromatography data have demonstrated that the hydrophobins HFBI and HFBII from Trichoderma reesei are aggregated in solution prior to ordered self-assembly.29 Because the fluorescence probe bis-ANS is known to bind nonpolar cavities or hydrophobic patches on the exterior surface of proteins with high affinity,30 the surface hydrophobicity of U-Sc3 molecules was examined by bis-ANS fluorescence. The fluorescence probe showed a very low affinity for U-Sc3 in solution, the observed fluorescence


Figure 6. Steady-state fluorescence of the probe Bis-ANS in the presence of Sc3: (A) bis-ANS emission spectra in the presence of U-Sc3, S-Sc3 (120 h), and I-Sc3 aliquots; (B) bis-ANS fluorescence intensity at λ ) 525 nm in the presence of S-Sc3 aliquots over a period of 120 h.

being only slightly above that of bis-ANS in buffer alone (Figure 6A). This indicates either that very few binding sites (hydrophobic patches) are present on the surfaces of the unimeric Sc3 molecules or that Sc3 is present in the form of micelles or other associations in which the hydrophobic portions of the individual molecules are shielded from water. In view of the predominance of nonpolar amino acids in the primary structure of Sc3, the existence of loosely associated structures seems the more likely scenario. The fluorescence probe ThT, known for its specific ability to interact with stacked β-sheet structures present in amyloid fibrils,27 did not bind to U-Sc3 (Figure 7A,B). Although large amounts of β-sheet structure have been reported previously to exist in unassembled Sc31,10,28,31 and other class I hydrophobins,5,18 the low fluorescence intensity of ThT indicates that the secondary structure under these conditions does not consist of β-sheet stacks. U-Sc3 Assembly into I-Sc3 Complexes at the Interface. The U-Sc3 solution that had been subjected to vortexing immediately assembled into SDS-insoluble structures (I-Sc3). This was shown by the complete disappearance of the 24 kDa band, associated with U-Sc3, in gels (Figure 1, panel A, lane 2, and panel D) and by the large percentage (82%) of protein that sedimented into a pellet after centrifugation of the vortexed solution (Figure 2). Presumably, U-Sc3 selfassembles at the air/water interface forming an SDS-stable, 10 nm amphipathic coating around the air bubbles created by vortexing, as has been previously reported by Wo¨sten et


al.6 The spherical Sc3-coated air bubbles, visualized by light microscopy, varied in size and reached up to 200 µm in diameter (Figure 4B). Density gradient centrifugation confirmed quantitative assembly into I-Sc3 structures of variable size, because Sc3 was present throughout the sucrose gradient (Figure 3C). These irreversible I-Sc3 complexes were too large to enter the polyacrylamide gel. I-Sc3 structures showed a high affinity for bis-ANS, as demonstrated by a 5-fold increase in the maximum fluorescence intensity and a blue shift in the emission wavelength maximum as compared to the U-Sc3 sample (Figure 6A). The transition from U-Sc3 to I-Sc3 apparently involves orientation (templating) of the protein at the air/water interface that is accompanied by increased exposure of hydrophobic residues on the I-Sc3 surface. As reported by de Vocht et al.,10 this transition may also involve a conformational change permitting an eventual increase in β-sheet content and β-sheet stacking, as indicated by ThT fluorescence (Figure 7A). The fluorescence yield in the experiments reported here is substantially higher than that previously reported21 because of an improved protocol of handling and treatment of Sc3 after purification. In addition, Congo Red has also been shown to interact with I-Sc3 in solution in a manner consistent with the existence of stacked β-sheet structures.18,21,32 U-Sc3 Assembly into S-Sc3 Complexes. A solution of U-Sc3 that was allowed to stand undisturbed (condition 2) was also shown to assemble into SDS-insoluble structures (S-Sc3) but over a considerable period of time. This type of assembly has not been previously reported or studied in detail to our knowledge. The slow appearance of S-Sc3 structures with time was followed by diminished intensity of the 24 kDa band in aliquots of the solutions taken in temporal order (Figure 1, panel A, lanes 4-8, and panel D). The decrease in the 24 kDa band intensity indicated that unimeric Sc3 assembled into S-Sc3 structures, which are not only large but also stable in the presence of SDS. The gradual formation of S-Sc3 complexes is also supported by the increase in protein concentration in the pellets and the concomitant decrease in the supernatant of centrifuged aliquots (Figure 2). In addition, the slow assembly of U-Sc3 into S-Sc3 was demonstrated by AFM observation of globular structures (20-200 nm) that increased in number and size over the 120 h period (Figure 5B,C). Some S-Sc3 structures were large enough to be observed by light microscopy or even with the naked eye. These aggregates were irregularly shaped and polydisperse (Figure 4A). In contrast to I-Sc3 assembly, which appears to be nearly quantitative and immediate at high concentrations, S-Sc3 structures assemble slowly over time and therefore coexist with U-Sc3. This coexistence was confirmed by simultaneously observing U-Sc3 in fractions near the top of the sucrose gradient and SDS-insoluble S-Sc3 in fractions near or at the bottom (Figure 3B). In addition, AFM indicated the presence of both globule-like structures and 10 nm rodlets for U-Sc3 sampled under condition 2 over time and dried onto mica (Figure 5). The rodlet pattern is presumed to originate from U-Sc3 that was dried onto the mica, whereas the globular species are representative of the S-Sc3 structures.

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Figure 7. The fluorescence of ThT in the presence of aliquots taken from (A) U-Sc3 and I-Sc3 samples, (B) U-Sc3 sample and buffer alone, (C) S-Sc3 samples taken over time (72 and 120 h), and (D) vortexed Sc3 samples demonstrating conversion from S-Sc3 to I-Sc3.

The fluorescence of ThT in the presence of S-Sc3 does not significantly change over 120 h, indicating that Sc3 complex formation under condition 2 occurs by a mechanism that does not involve β-sheet stacking (Figure 7A,C). Also, Congo Red does not interact with S-Sc3 assemblages in a manner consistent with the presence of β-sheet stacks. Although the S-Sc3 and I-Sc3 aggregates are SDS-insoluble, ThT fluorescence measurements and interactions with Congo Red indicate that S-Sc3 structures differ from those of I-Sc3 aggregates. S-Sc3 appears to be less-structured than I-Sc3, at least in terms of β-sheet stacking. Interestingly, S-Sc3 can be, at least partially, converted to I-Sc3 by vortexing, which clearly induces β-sheet stacking (Figure 7D and Figure 10). Whether the energy of vortexing or the microbubble surfaces or both induce an S-Sc3-to-I-Sc3 transition directly or whether S-Sc3 is converted to U-Sc3 and then to I-Sc3 is presently unknown. The Effect of Preassembled Sc3 “Seed” on S-Sc3 Formation. Many proteins associated with Alzheimer’s, Parkinson’s, and prion-related diseases self-assemble into insoluble, stacked β-sheet fibrils.19,20,33-35 The growth of amyloid fibrils is initiated by a chance misfolding of a single, or a few, protein molecules. These misfolded proteins apparently recruit and nucleate others, thus extending the amyloid fibril.19,20 Normally, higher concentration of amy-

loidogenic protein in solution results in higher probability for the nucleation event to occur and faster fibrillar growth.19,20 It is known that preassembled amyloid fibrils, when added to a solution of unassembled protein, act as seeds or nucleating sites for fibrillogenesis. With seeds, the amyloid assembly that normally occurs over several days can occur within hours or minutes.19,20 Various forms of assembled Sc3 were added to U-Sc3 to test the effect of seeding on the rate of S-Sc3 assembly. None of the three types of “seed” added to the U-Sc3 solutions enhanced the rate of S-Sc3 self-assembly; in fact, the presence of seed appears to be inhibitory (Figure 8). This inhibition is in direct contrast to the kinetics of seeded fibril formation observed for other amyloidogenic proteins. However, it is consistent with the ThT fluorescence (Figure 7) and Congo Red absorption data (not shown) that indicate S-Sc3 does not assemble in a nucleated fashion and does not form stacked β-sheets. Class I Hydrophobin Films. Class I hydrophobins, including Sc3, have been shown to assemble into amphipathic films at hydrophilic/hydrophobic interfaces, including the fungal surface.6 When the amphipathic layer is visualized from the hydrophobic side with transmission electron microscopy (TEM) or atomic force microscopy (AFM), a rodlet pattern can be observed.3,5 Also, if an unassembled



Figure 8. The effect of preassembled Sc3 (seeds) on the rate of U-Sc3 assembly into S-Sc3 structures. Assembly of Sc3 was monitored with SDS-PAGE by the decrease in the 24 kDa band (A) with and without 2.5 µg of I-Sc3 seed and (B) with and without 5.0 µg of I-Sc3 seed. Quantitative densitometry of the 24 kDa bands after SDS-PAGE of (C) I-Sc3 seeded and control samples over 120 h, (D) S-Sc3 seeded and control samples over 120 h, and (E) dried Sc3 seeded and control samples over 120 h is also shown.

Figure 9. The effect of U-Sc3 concentration on the assembly of U-Sc3 into S-Sc3 and I-Sc3 complexes: (A) quantitative densitometry of 24 kDa bands monitoring S-Sc3 assembly after SDS-PAGE analysis; (B) the effect of concentration of the percentage on U-Sc3 assembled into S-Sc3 and I-Sc3 structures.

Sc3 solution is dried onto a hydrophilic surface such as mica, the same rodlet pattern can be observed covering the hydrophilic substrate (Figure 5A).1,10,18,36 This has been a trademark of class I hydrophobins. How the resulting film assembles at the interface and whether surface-induced ordering is the same in the absence of vortexing forces is unknown. Rodlets have never been shown to exist in solution, and in this study, the addition of assembled Sc3 to the U-Sc3 solutions did not increase the rate of Sc3 self-assembly. Furthermore, class II hydrophobins, which assemble into films similar to those of class I hydrophobins, do not display the characteristic rodlet morphology on either side of the film.5,9 Class I hydrophobins possess characteristics common to amyloid fibrils, such as β-sheet stacking,18,21 and have been compared to the fibrils of amyloidogenic proteins. Despite these similarities, rodlets can only be seen from one side of the assembled hydrophobin film, indicating that they are not cylindrical and are thus unlike amyloid fibrils.4 AFM has also shown that rodlets appear to be untwisted and much shorter than amyloid fibrils, further supporting that these two types of assembled structures have different appearances and may assemble by quite different mechanisms.1,18 Assembly of Sc3 in aqueous solution was shown to be independent of seeding, in contrast to that of amyloidogenic proteins.20,35 Instead, in I-Sc3 assembly, the interface appears to induce ordering. The folded Sc3 chain has a high propensity to migrate to air/water,6,10,31 oil/water,6,13,37 and

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Figure 10. Model of Sc3 Assembly into S-Sc3 and I-Sc3 structures from a U-Sc3 solution.

hydrophobic solid/water10,13 interfaces spontaneously. The amphipathic Sc3 molecules may preferentially align at the interface and undergo the conformational change that has been previously reported10,28 to result in the formation of a film of approximately 10 nm thick. The rodlet pattern can be visualized from the hydrophobic side of the coating31 once the film has reached its final assembled state at the various hydrophobic/hydrophilic interfaces. In the absence of the proper alignment or templating caused by the interface, I-Sc3 assembly cannot occur. It appears that the S-Sc3 type of association can only occur in the absence of an interface and other interfering agents (TFA, the schizophyllan polysaccharide). Concentration Dependence of I-Sc3 and S-Sc3 Assembly. The percentage of assembled I-Sc3 after vortexing increases with increasing concentration of U-Sc3 (Figure 9B). This can be explained by an increase in the portion of the interfacial surface (air bubbles) that is coated with U-Sc3. If the U-Sc3 concentration is high enough, intermolecular interactions between Sc3 molecules aligned at the interface result in the formation of a membrane or film. If the protein concentration is low and the interfacial surface area is large, the Sc3 protein cannot assemble into a two-dimensional film because an insufficient number of Sc3 molecules may be harbored by individual air microbubbles. This critical concentration requirement for Sc3 assembly into an amphipathic film has been suggested previously.5 At present, we are unable to explain the mechanism and unexpected concentration dependence of S-Sc3 assembly from aqueous solutions under quiescent conditions. A structure other than that of stacked β-sheets is formed, probably one in which hydrophobic, hydrogen bonding, and polar interactions “drive” assembly but still allow sufficient dispersion by steric stabilization to preclude early complex

precipitation. Such structures are apparently not formed in the presence of sufficient quantities of the polysaccharide, schizophyllan, and of course, TFA. Some small classical and polymeric surfactants form higher-ordered structures, such as micelle-to-hexagonal phase transitions, with an increase in concentration. This does not seem to be the case in S-Sc3 aggregation. The peculiar inhibitory effect on S-Sc3 nucleation by a preassembled S-Sc3 “seed” is remarkable for associating amphiphilic molecules and needs further study. It is clear that I-Sc3 “seed” added to the U-Sc3 solution does not induce further I-Sc3 formation in the form of insoluble crystals or amyloid-like fibers. Clearly, association into the I-Sc3 state requires orientation at an interface and (probably) sufficient energy for assembly. Conclusions Evidence has been presented confirming that, depending on experimental conditions, U-Sc3 assembles from aqueous solutions into two distinct forms with totally different characteristics. Under condition 1, in which both an air/water interface and sufficient energy are provided, interfacial templated assembly occurs. The resulting I-Sc3 structures are SDS-insoluble, and are comprised of β-stacked sheets. Under condition 2, a time-dependent self-assembly from a quiescent solution in the absence of an air/water interface results in S-Sc3 structures. Techniques including fluorescence spectroscopy, SDS-PAGE, density gradient centrifugation, and phase contrast and atomic force microscopy have been utilized to compare and contrast the characteristics of the S-Sc3 and I-Sc3 states, as well as to discern solution concentrations of unimeric Sc3 during the respective assembly processes. A model based on this work and reports in the literature is proposed (Figure 10) that postulates three


distinct types of Sc3 structures: (1) soluble Sc3 consisting of unimers or multimers in loose micelle-like associations, (2) an interfacially assembled I-Sc3 with extensive β-sheet stacking occurring under condition 1, and (3) a solutionassembled S-Sc3 resulting from a time-dependent ordering in aqueous media in the absence of a template and without external perturbation. While the formation of I-Sc3, also called the β-sheet state,5,10,28,31 has been thoroughly investigated, assembly of Sc3 into the form that we have designated S-Sc3 has not been observed to our knowledge. We have reported previously14,17 the role of the cosecreted fungal polysaccharide, schizophyllan, in inhibiting aggregation of Sc3 in aqueous solutions. Now a major challenge will be to elucidate the precise mode of solution assembly in the absence of an interface and of inhibitory molecules. It will be of considerable interest to determine whether the peculiar inverse concentration dependence of S-Sc3 aggregation is a result of natural regulatory design or simply an artifact of in vitro conditions. Acknowledgment. The authors gratefully acknowledge the Office of Naval Research, Geltex Pharmaceuticals, and the MERSEC program of the National Science Foundation (Grant DMR-0213883) for support of this research. References and Notes (1) de Vocht, M. L.; Reviakine, I.; Wo¨sten, H. A.; Brisson, A.; Wessels, J. G.; Robillard, G. T. J. Biol. Chem. 2000, 275, 28428-28432. (2) Wessels, J. G. H. Annu. ReV. Phytopathol. 1994, 32, 413-437. (3) Wessels, J. G. AdV. Microb. Physiol. 1997, 38, 1-45. (4) Wo¨sten, H. A. Annu. ReV. Microbiol. 2001, 55, 625-646. (5) Wo¨sten, H. A.; de Vocht, M. L. Biochim. Biophys. Acta 2000, 1469, 79-86. (6) Wo¨sten, H. A. B.; de Vries, O. M. H.; Wessels, J. G. H. Plant Cell 1993, 5, 1567-1574. (7) Russo, P. S.; Blum, F. D.; Ipsen, J. D.; Yusuf, J. A.; Miller, W. G. Can. J. Bot. 1982, 60, 1414-1422. (8) Scholtmeijer, K.; Wessels, J. G. H.; Wo¨sten, H. A. B. Appl. Microbiol. Biotechnol. 2001, 56, 1-8. (9) Wessels, J. G. Fungal Genet. Biol. 1999, 27, 134-145. (10) de Vocht, M. L.; Scholtmeijer, K.; van der Vegte, E. W.; de Vries, O. M.; Sonveaux, N.; Wo¨sten, H. A.; Ruysschaert, J. M.; Hadziloannou, G.; Wessels, J. G.; Robillard, G. T. Biophys. J. 1998, 74, 2059-2068. (11) Wo¨sten, H. A.; Schuren, F. H.; Wessels, J. G. EMBO J. 1994, 13, 5848-5854. (12) Goodwin, J. S.; McCormick, C. L.; Cannon, G. C. Unpublished results.

Biomacromolecules, Vol. 4, No. 4, 2003 967 (13) Wo¨sten, H. A. B.; Ruardy, T. G.; van der Mei, H. C.; Busscher, H. J.; Wessels, J. G. H. Colloids Surf. B 1995, 5, 189-195. (14) Martin, G. G.; Cannon, G. C.; McCormick, C. L. Biopolymers 1999, 49, 621-633. (15) Wo¨sten, H. A.; van Wetter, M. A.; Lugones, L. G.; van der Mei, H. C.; Busscher, H. J.; Wessels, J. G. Curr. Biol. 1999, 9, 85-88. (16) Wo¨sten, H. A.; de Vries, O. M.; van der Mei, H. C.; Busscher, H. J.; Wessels, J. G. J. Bacteriol. 1994, 176, 7085-7086. (17) Martin, G. G.; Cannon, G. C.; McCormick, C. L. Biomacromolecules 2000, 1, 49-60. (18) Mackay, J. P.; Matthews, J. M.; Winefield, R. D.; Mackay, L. G.; Haverkamp, R. G.; Templeton, M. D. Structure 2001, 9, 83-91. (19) Garzon-Rodriquez, W.; Vega, A.; Sepulveda-Becerra, M.; Milton, S.; Johnson, D. A.; Yatsimirsky, A. K.; Glabe, C. G. J. Biol. Chem. 2000, 275, 22645-22649. (20) Serio, T. R.; Cashikar, A. G.; Kowal, A. S.; Sawicki, G. J.; Moslehi, J. J.; Serpell, L.; Arnsdorf, M. F.; Lindquist, S. L. Science 2000, 289, 1317-1321. (21) Butko, P.; Buford, J. P.; Goodwin, J. S.; Stroud, P. A.; McCormick, C. L.; Cannon, G. C. Biochem. Biophys. Res. Commun. 2001, 280, 212-215. (22) Kelly, J. W. Structure 1997, 5, 595-600. (23) Blake, C.; Serpell, L. Structure 1996, 4, 989-997. (24) Hatters, D. M.; MacPhee, C. E.; Lawrence, L. J.; Sawyer, W. H.; Howlett, G. J. Biochemistry 2000, 39, 8276-8283. (25) Dons, J. J.; de Vries, O. M.; Wessels, J. G. Biochim. Biophys. Acta 1979, 563, 100-112. (26) Laemmli, U. K. Nature 1970, 227, 680-685. (27) LeVine, H., III Methods Enzymol. 1999, 309, 274-284. (28) Wang, X.; Vocht, M. L.; Jonge, J. D.; Poolman, B.; Robillard, G. T. Protein Sci. 2002, 11, 1172-1181. (29) Torkkeli, M.; Serimaa, R.; Olli, I.; Linder, M. Biophys. J. 2002, 83, 2240-2247. (30) Mazumdar, M.; Parrack, P. K.; Mukhopadhyay, K.; Bhattacharyya, B. Biochemistry 1992, 31, 6470-6474. (31) de Vocht, M. L.; Reviakine, I.; Ulrich, W. P.; Bergsma-Schutter, W.; Wo¨sten, H. A.; Vogel, H.; Brisson, A.; Wessels, J. G.; Robillard, G. T. Protein Sci. 2002, 11, 1199-1205. (32) Klunk, W. E.; Jacob, R. F.; Mason, R. P. Methods Enzymol. 1999, 309, 285-305. (33) Fandrich, M.; Fletcher, M.; Dobson, C. Nature 2001, 410, 165166. (34) Balbirnie, M.; Grothe, R.; Eisenberg, D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2375-2380. (35) Serpell, L. Biochim. Biophys. Acta 2000, 1502, 16-30. (36) Gunning, A. P.; De Groot, P. W. J.; Visser, J.; Morris, V. J. J. Colloid Interface Sci. 1998, 201, 118-126. (37) Zangi, R.; De Vocht, M. L.; Robillard, G. T.; Mark, A. E. Biophys. J. 2002, 83, 112-124. (38) Lugones, L. G.; Wo¨sten, H. A. B.; Birkenkramp, K. U.; Sjollema, K. A.; Zagers, J.; Wessels, J. G. H. Mycol. Res. 1999, 103, 635-640.

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Experimental Evidence for Multiple Assembled States of Sc3 from

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