Amyloid Fibril Formation by Peptide LYS (11-36) in Aqueous

Antonio N. Calabrese , Yanqin Liu , Tianfang Wang , Ian F. Musgrave , Tara L. Pukala , Rico F. Tabor , Lisandra L. Martin , John A. Carver , John H. B...
0 downloads 0 Views 194KB Size
Download PDF
Biomacromolecules 2004, 5, 1818-1823

1818

Amyloid Fibril Formation by Peptide LYS (11-36) in Aqueous Trifluoroethanol Wei Liu,† John M. Prausnitz,†,‡ and Harvey W. Blanch*,† Chemical Engineering Department, University of California, Berkeley, Berkeley, California 94720, Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received March 17, 2004; Revised Manuscript Received June 23, 2004

Peptide LYS (11-36), derived from the β-sheet region of T4 lysozyme, forms an amyloid fibril in aqueous trifluoroethanol (TFE) at elevated temperature. The peptide has a moderate R-helix content in 20 and 50% (v/v) TFE solution; large quantities of fibrils were formed after incubation at 55 °C for 2 weeks as monitored by a thioflavin T fluorescence assay. No fibrils were observed when the peptide initially existed predominantly as a random coil or as a complete R helix. Our results suggest that a moderate amount of R helix and random coil present in the peptide initially facilitates the fibril-formation process, but a high R-helix content inhibits fibril formation. Transmission electron microscopy revealed several types of fibril morphologies at different TFE concentrations. The fibrils were highly twisted and consisted of interleaved protofilaments in 50% TFE, while smooth and flat ribbonlike fibrils were found in 20% TFE. In 50% TFE, the fibril growth rate of LYS (11-36) was found to depend strongly on peptide concentration and seeding but was insensitive to solution pH and ionic strength. Introduction Protein aggregation is ubiquitous in the production and formulation of therapeutic proteins and is the probable cause of a number of neurodegenerative diseases. Aggregated proteins have structures that vary from amorphous to highly ordered β-sheet aggregates. One of the most important examples of a highly ordered aggregate is the insoluble amyloid fibril that has been found to be associated with several debilitating diseases including Alzheimer’s, Parkinson’s, and Huntington’s diseases.1,2 To date, nearly 20 such disease-causing proteins have been identified in humans. Although these proteins have no sequence homology and are structurally diverse, amyloid fibrils from all of them share a characteristic cross-β-sheet structure. Recently, it has been shown that amyloid fibril formation is not limited to proteins associated with diseases, which has led to the view that amyloid fibril formation is a common property of all proteins under appropriate conditions.3-5 The formation of fibrils may be a fundamental property of the polypeptide backbone. Therefore, a study of amyloid fibril formation from nondisease-associated proteins may provide useful information to elucidate the mechanism for amyloid fibrillogenesis. Globular proteins may partially denature or unfold prior to forming fibrils.6,7 Under denaturing conditions, native structures are largely disrupted, while a certain amount of secondary structure remains. Upon forming fibrils, the protein attains an extensive β-sheet structure that is stabilized by * To whom correspondence should be addressed. Address: 201 Gilman Hall, Department of Chemical Engineering, University of California, Berkeley, Berkeley, CA 94720-1462. Telephone: (510) 642-1387. Fax: (510) 643-1228. E-mail: [email protected]. † University of California, Berkeley. ‡ Lawrence Berkeley National Laboratory.

intermolecular hydrogen bonds and hydrophobic and electrostatic interactions.8-11 Solution conditions for fibril formation include the presence of cosolvents, elevated temperature, and extreme pH.12-14 In contrast to globular proteins, polypeptides have higher conformational flexibility and amyloid fibrils can be formed by peptides consisting primarily of random coils or partially ordered secondary structures.15,16 Despite recent research on amyloid fibrillogenesis, we have only a poor understanding of the molecular mechanism of fibril formation and the role of peptide structure, particularly in the early stage of fibril formation. To explore the role of peptide conformation and solution conditions on amyloid fibril formation, we studied peptide LYS (11-36) fibrillogenesis in aqueous trifluoroethanol (TFE) solutions. Because under some circumstances the presence of TFE mimics the properties of a biological membrane,17,18 our study may help to shed light on protein amyloid fibril formation in a membrane environment. Several variants of lysozyme provide a model for amyloid fibrillogenesis. Destabilized hen egg-white lysozyme12,13 and human lysozyme mutants19 are capable of undergoing amyloid fibrillogenesis. Two peptides derived from the β-sheet region of hen lysozyme were also found to form fibrils readily, and the β domain was suggested to be of particular significance in the formation of fibrils from the full-length protein.13 T4 lysozyme consists of eight helices and one antiparallel β-sheet region (Figure 1);20,21 it has not been found associated with any diseases. Peptide LYS (1136) corresponds to the β-sheet region of T4 lysozyme and has been identified as one of the initial protein folding sites.23 Recent studies have shown that LYS (11-36) has a selfassociation propensity, and the strength of self-association increases with pH and the addition of TFE.24,25 Thus, LYS

10.1021/bm049841e CCC: $27.50 © 2004 American Chemical Society Published on Web 07/30/2004

Amyloid Fibril Formation of a Peptide

Figure 1. X-ray crystal structure of T4 lysozyme.20,21 Peptide LYS (11-36) corresponding to the β-sheet region of T4 lysozyme has been highlighted. The diagram was created using RasMol.22

(11-36) provides a good model to study the effect of peptide conformation and solution conditions on amyloid fibril formation. In the present work, we converted peptide LYS (11-36) into amyloid fibrils in TFE aqueous solution. We also studied the effect of environmental factors (pH, ionic strength, peptide concentration, and seeding) on the kinetics of fibril formation. Materials and Methods TFE and Thioflavin T (ThT) were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium chloride, sodium phosphate, NaOH, and HCl were purchased from Fisher Scientific Co. (Pittsburgh, PA). Deionized water was obtained from a Barnested Nanopure II filtration system. Peptide Synthesis. Peptide LYS (11-36) has the sequence EGLRLKIYKDTEGYYTIGIGHLLTKS. It was synthesized by solid-phase methodology and purified by reverse-phase high-performance liquid chromatography. The peptide has a theoretical isoelectric point of 9.9 and molecular weight of 2969.44 Da. Electrospray ionization mass spectrometry yielded a molecular weight of 2969.40 Da. Preparation of Peptide Solutions. Peptide stock solution (∼2 mM) was prepared by dissolving the lyophilized peptide powder in water and then filtering through a 0.2-µm poresize Anotop inorganic syringe filter (Whatman). The peptide concentration was determined using a Beckman DU 640 spectrophotometer with an extinction coefficient of 3840 M/cm. The pH of peptide aqueous solutions was measured to be 4 ( 0.1. The addition of TFE did not change the peptide solution pH significantly. No adjustment of pH was made except for the particular experiments to determine the effect of pH on peptide conformation and fibril formation, where small aliquots of NaOH and HCl were used. Under each solution condition, 2 mL of peptide solution was incubated at the desired temperature, and 10-µL aliquots of peptide solution were drawn periodically for the ThT assay. The peptide concentration was 30 µM for most experiments except for the particular experiments to determine the effect of the peptide concentration on fibril formation, where the initial peptide concentrations were 210, 3, and 0.3 µM, respectively. In the seeding experiment, preformed peptide fibrils (initial peptide concentration: 30 µM) in 50% TFE were used as seeds. Aliquots of 20, 100, and 210 µL of preformed fibrils were added to 2 mL of fresh peptide solution (30 µM) such that the mixed solution contained 1, 5, or 10% (v/v) seeds, respectively. ThT Assay. ThT stock solution (1 mM) was prepared in 10 mM phosphate buffer with 150 mM NaCl at pH 7 and

Biomacromolecules, Vol. 5, No. 5, 2004 1819

kept dark at 4 °C. ThT working solution (50 µM) was filtered through a 0.2-µm filter before measurement. For each sample measurement, 10 µL of peptide solutions and 200 µL of ThT working solution were added to a 96-microwell plate. The fluorescence intensity of the mixed solution was read on a Tecan fluorometer (Tecan Group, Ltd., Switzerland) with excitation wavelength 450 nm (slit width 5 nm) and emission wavelength 482 nm (slit width 5 nm) at room temperature. Each reported result was obtained from an average of five measurements. Circular Dichroism (CD). Far-UV CD spectra for peptide and amyloid fibrils were obtained with an Aviv model 62DS CD spectropolarimeter using a 1-mm path-length cuvette. Peptide solutions (30 µM) were prepared in different TFE concentrations, and the spectra were recorded at 25, 37, and 55 °C. After incubation for 2 weeks, CD spectra were also recorded for the peptide solutions forming amyloid fibrils. Trace amounts of visible aggregates were removed by centrifuge. The wavelength range was 190-250 nm, with intervals of 0.5 nm and a bandwidth of 1.5 nm. Background spectra of water and the TFE solution, recorded in the absence of peptide, were subtracted from the sample spectra. Transmission Electron Microscopy (TEM). TEM images of samples were obtained with a Tecnai 12-120 kV transmission electron microscope (FEI Co., OR). The 5-µL peptide samples obtained after 4 days and 2 weeks of incubation were placed on a Formvar-coated grid and negatively stained with 2% (w/v) uranyl acetate in water, washed, air-dried, and then examined at an accelerating voltage of 80 kV. Results and Discussion Peptide Conformation in Aqueous Solutions of Different TFE Concentrations. Figure 2A shows the conformations of peptide LYS (11-36) in different TFE concentrations at 55 °C at pH 4. Identical CD spectra were found at 25 and 37 °C. The peptide spectra in water and 10% TFE showed a minimum around 197 nm indicating that the peptide attained a predominantly random-coil conformation. Upon increasing the TFE concentration to 20%, the minimum of the spectrum shifted to 209 nm and the peptide existed in solution with a small R-helical component even though mostly as random coil. The helix content increases continuously in the range of 20-80% TFE. In 80% TFE, the peptide has a complete R-helical structure. In 20% TFE solution, Najbar et al.26 found that peptide LYS (11-36) attains a β-sheet structure in contrast to the R helix found in our study. This discrepancy may have resulted from a difference in solution pH, salt type, or ionic strength. But more importantly, the different spectra indicate that the amount of structured peptide is low in 20% TFE. In native T4 lysozyme, LYS (11-36) exists as a β-sheet structure. Such β-sheet fold might be stabilized by many nonlocal contacts from surrounding side chains, which do not exist in the peptide fragment. Anderson et al.24 have observed a transition from random coil at pH 4 to β sheet at pH 7 for peptide LYS (11-36) in aqueous solution. However, in 50% TFE solution, we obtained identical R-helix CD spectra for peptide LYS

1820

Biomacromolecules, Vol. 5, No. 5, 2004

Figure 2. Far-UV CD spectra of peptide LYS (11-36). (A) Peptide conformation at different TFE concentrations; (B) peptide conformation in amyloid fibrils after 2 weeks of incubation in 20% (thick, dashed line) and 50% (thick, solid line) TFE. The initial peptide conformation is shown in thin lines for comparison. These spectra were recorded at 55 °C and pH 4.

Figure 3. Amyloid fibril formation of peptide LYS (11-36) at different TFE concentrations at 55 °C and pH 4, monitored by ThT fluorescence. The peptide concentration was 30 µM. Lines are to guide the eye.

(11-36) at pH 2, 4, 7, and 10. Thus, in 50% TFE, the peptide charge does not affect conformation, in contrast to the results observed in pure aqueous solution. Peptide Amyloid Fibril Formation in TFE Solutions. Peptide solutions were prepared in aqueous 0, 10, 20, 50, and 80% (v/v) TFE and incubated at 25, 37, and 55 °C. At 25 and 37 °C, no amyloid fibrils were found after incubation for 1 month. However, at 55 °C, large quantities of fibrils were found in 20 and 50% TFE solutions. The formation of amyloid fibrils was monitored by a ThT fluorescence assay. ThT can associate rapidly with aggregated amyloid fibrils, shifting the excitation maximum (ex) of 385 nm and emission maximum (em) of 445 nm of the free dye to 450 nm (ex) and 482 nm (em) for the binding complex.27,28 Figure 3 shows the growth of peptide fibrils as observed by an increase in fluorescence intensity measured at 482 nm. No fibrils were observed in 0, 10, and 80% TFE solutions held for up to 1 month at 55 °C. Upon forming fibrils, the peptide solution

Liu et al.

becomes a slightly viscous clear gel. ThT has also been known to bind to nonfibril forms of peptide aggregates.29 Thus, far-UV CD was used to determine the peptide conformation in aggregated states and TEM was used to visualize the fibrils. The CD spectra of fibril solutions in 20 and 50% TFE were recorded, and a minimum at 216 nm, a typical β-sheet structure, was found for both concentrations. Figure 2B shows the peptide conformation transition from R helix in initial solution to β sheet upon forming amyloid fibrils. In previous studies, amyloid fibrils have been found formed by polypeptides adopting different structures, varying from predominantly random coil to primarily R helix.15,16 However, in the present study, peptide LYS (11-36) was not able to form amyloid fibrils when present as a random coil in aqueous solution. Upon addition of TFE, the peptide adopts an increasing amount of R-helical structure and amyloid fibrils were formed in 20 and 50% TFE solution. When the peptide is in a complete R-helix structure (80% TFE), the amyloid fibril formation was again inhibited. From these observations, we conclude that the relative population of structured and nonstructured conformations, which in this case depends on the percentage of TFE, plays an important role in the formation of amyloid fibrils from this peptide in particular, and it might be the case for other peptides that are partially folded in solution. TFE is a widely used protein denaturant. Several proteins and polypeptides form fibrils in TFE solutions.4,13 TFE can bind to protein or peptide hydrophobic amino acid residues causing weaker inter- and intramolecular hydrophobic interactions.30-32 The weakened intramolecular hydrophobic interaction may lead to protein destabilization and unfolding, while reduced intermolecular hydrophobic interaction may decrease protein or peptide attractions.32 TFE also stabilizes peptide secondary structures (R helixes and β sheets) by affecting peptide hydrogen bonding. TFE is a slightly stronger proton donor than water, but it is a much weaker proton acceptor.33 Adding TFE to an aqueous solution significantly decreases the solvent’s ability to compete with peptide carbonyl acceptors. Thus, the peptide intra- and intermolecular hydrogen bonding abilities are strengthened by addition of TFE.34 The overall effect of TFE on polypeptide conformation and fibril formation can be explained by a balance effect between decreased hydrophobic interactions and increased hydrogen bonding: at low TFE concentration, peptide hydrogen bonding is too weak to allow formation of either inter- or intramolecular structure, and no partially ordered oligomeric intermediates could be formed; at high TFE concentrations, hydrophobic interactions are too weak to allow intermolecular association. At intermediate TFE concentrations, stabilization of hydrogen bonding is sufficient to balance the reduction in hydrophobic association, and highly structured β-sheet assemblies can be formed. The increase in intermolecular attraction for peptide LYS (1136) by adding an intermediate amount of TFE was also observed in the previous study using fluorescence anisotropy.25 Chiti et al.4 suggests that a TFE concentration higher than 35% does not favor and may even inhibit protein aggregation.

Amyloid Fibril Formation of a Peptide

Figure 4. Electron micrograph of negatively stained amyloid fibrils formed in 20% TFE solution at pH 4. (A) Short fibrils formed after 4 days of incubation at 55 °C; (B) mature fibrils after 2 weeks. Box 1 shows the association of 4 strands of protofilaments to form a flat ribbonlike fibril. Box 2 shows a curved fibril. The peptide concentration was 30 µM. (Bar ) 100 nm.)

MacPhee and Dobson8 also reported that 50% TFE could dissociate preformed fibrils assembled by a 10-residue peptide. However, the peptide LYS (11-36) forms wellstructured fibrils in 50% TFE solution, suggesting that the effect of TFE on amyloid fibril formation depends primarily on whether a peptide can adopt a stable secondary structure in the presence of TFE. For most polypeptides, helicity is at a maximum at 35% TFE.35 LYS (11-36) has a low propensity to adopt secondary structures and only attains a small R-helix content in 20% TFE and a moderate of R-helix content in 50% TFE. In 80% TFE, the peptide is present as a complete R helix, and fibril formation is inhibited. Our results suggest that TFE can either facilitate or inhibit protein fibril formation depending on its concentration, and such concentration dependence varies significantly among amino acid sequences. The peptide amyloid fibrillogenesis may depend primarily on the inherent structural propensity and hydrogen bonding ability of the peptide; amyloid fibrils are formed in a highly concentrated TFE solution if a moderate amount of R-helix structure is present. Morphologies of Amyloid Fibrils. The fibrils were observed by TEM. In both 20 and 50% TFE solution, short fibrils or protofibrils were found after 4 days of incubation (Figures 4A and 5A) and long fibrils were found after 2 weeks of incubation (Figures 4B and 5B). The fibrils formed in 50% TFE solution contain a much denser fibril network compared to those in 20% TFE solution. In both solutions, the assembly of fibrils consists of several single protofilaments. However, the characteristics of individual protofilaments and the morphology of the amyloid fibrils were different. In 20% TFE, the single protofilament has a

Biomacromolecules, Vol. 5, No. 5, 2004 1821

Figure 5. Electron micrograph of negatively stained amyloid fibrils formed in 50% TFE solution at pH 4. (A) Short fibrils formed after 4 days of incubation at 55 °C; (B) mature fibrils after 2 weeks. Box 1 shows two smooth tubular fibrils. Box 2 shows several highly twisted protofilaments forming large fibrils. The peptide concentration was 30 µM. (Bar ) 100 nm.)

diameter of about 4 nm and the amyloid fibrils consist of four or five individual protofilaments. Most of the fibrils are smooth and have a flat, ribbonlike morphology. The shapes of the fibrils are highly curved; occasionally, loop structures were found. Fibrils with such a high degree of curvature are not unusual; they have also been observed with insulin fibrils.36 In 50% TFE, however, the single protofilaments have a larger diameter, about 8 nm. The fibrils are straight and composed of many highly twisted individual protofilaments. Assembly of peptides into protofilaments may involve both electrostatic and hydrophobic forces, while the assembly of protofilaments into fibrils involves predominantly weak hydrophobic forces.8,9,37 Thus, the difference in initial peptide conformations may lead to different fibril intermediates responsible for the fibril morphology difference. Self-assembly of amyloid-forming proteins may be used to form template nanomaterials.38-40 For design of fibril arrays and highly regular nanotubes, it is useful to explore environmental conditions that affect fibril self-assembly and fibril morphology. The difference in morphology of amyloid fibrils observed at different TFE concentrations suggests that control of solution composition may provide a route for the assembly of a particular desired fibril array for highly regular nanotubes. Effect of Environmental Factors on Fibril-Forming Kinetics. Effect of Electrostatic Interactions. We investigated the fibril formation of the peptide at pH values of 2, 7, and 10 in 50% TFE at 55 °C. The ThT fluorescence assay showed that all solutions formed fibrils, and the rates of fibril formation were essentially the same as that at pH 4. No fibrils

1822

Biomacromolecules, Vol. 5, No. 5, 2004

Figure 6. Effect of peptide concentration on the kinetics of fibril formation. Initial peptide concentrations were 0.3, 3, 30, and 210 µM, respectively. The peptide solution was 50% TFE at pH 4, 55 °C. Lines are to guide the eye.

were observed in solutions at all pHs in the absence of TFE. For the 50% TFE solutions at pH 4, fibril formation kinetics was determined in the presence of 0.2 and 0.5 M NaCl. Both solutions formed fibrils, but fibril growth rates were independent of NaCl concentration. Many proteins form amyloid fibrils at a certain pH; maximum fibril formation occurs near the isoelectric point, where the protein is expected to be least soluble and electrostatic repulsion is weak.41 Nielsen et al.14 reported that increasing ionic strength may shorten the lag time and slow the growth rate of insulin fibrils. Several peptide β-sheet selfassemblies were also found to be influenced by electrostatic interactions.11,37 However, the present study shows that the fibril growth kinetics for LYS (11-36) is not affected by either the solution pH or the ionic strength in 50% TFE solution. The reason for the insensitivity of the LYS (1136) fibril growth rate on the electrostatic interactions is not clear. However, considering the identical R-helix CD spectra observed for peptide LYS (11-36) in 50% TFE at different pHs, we conclude that the amyloid fibril formation of LYS (11-36) is controlled more by intermolecular hydrogen bonding, hydrophobic interactions, and peptide conformation rather than by electrostatics. Effect of Peptide Concentration. Figure 6 illustrates the effect of peptide concentration on the kinetics of fibril formation in 50% TFE. In a 210 µM peptide solution, amyloid fibril was formed after incubating for about 50 h and reached full length after 100 h. With decreasing peptide concentrations, the rate of fibril formation decreased and exhibited a longer lag time. In solutions with a peptide concentration below 0.3 µM, no fibrils were observed. The highly concentration-dependent amyloid fibril formation suggests that partially stable oligomers may initially be present as a function of peptide concentration and eventually form highly ordered amyloid fibrils. Effect of Seeding. Figure 7 shows the effect of seeding on peptide fibril formation. The lag time decreased upon addition of 1 and 5% preformed fibril seeds to the peptide solution. With seed addition of 10%, the lag time completely disappeared. Fibrils were found to grow faster in solutions with seeds, as a result of the facilitated fibril nucleation process. The high dependence of the fibril-formation rate on fibril seeds and peptide concentration suggests that peptide LYS (11-36) fibril formation is nucleation-dependent.

Liu et al.

Figure 7. Effect of seeding on the kinetics of peptide fibril formation. Peptide concentration was 30 µM, and TFE concentration is 50%. The solution was at pH 4, 55 °C. All preformed fibril seeds were obtained under the same experimental conditions. Lines are to guide the eye.

Effect of Temperature. We found that LYS (11-36) forms fibrils only at a high temperature (55 °C). Such a temperature dependency might indicate that hydrophobic effects are important in fibril assembly. Conclusion Peptide LYS (11-36) derived from T4 lysozyme can form amyloid fibrils in aqueous TFE solution. TFE concentration and polypeptide conformation are found to be important for the fibril formation of the peptide. A moderate content of R helix and a percentage of random coils in the initial peptide solution may favor formation of partially structured intermediates with subsequent fibril formation, while a stable R helix inhibits fibril formation. Our results support Dobson and co-workers’ hypothesis3-5 that amyloid fibrillogenesis is a common generic property of all proteins and polypeptides. Acknowledgment. This work was supported by the National Science Foundation under Grant BES-0118208 and by the Office of Basic Energy Sciences of the U.S. Department of Energy. We thank Dr. David King for peptide synthesis and purification. The authors are grateful to Professor Susan Marqusee for the use of a CD spectropolarimeter. References and Notes (1) Lansbury, P. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3342. (2) Perutz, M. F. Trends Biochem. Sci. 1999, 24, 58. (3) Guijarro, J. I.; Sunde, M.; Jones, J. A.; Campbell, I. D.; Dobson, C. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4224. (4) Chiti, F.; Webster, P.; Taddei, N.; Clark, A.; Stefani, M.; Ramponi, G.; Dobson, C. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3590. (5) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329. (6) Kelly, J. W. Curr. Opin. Struct. Biol. 1996, 6, 11. (7) Kelly, J. W. Curr. Opin. Struct. Biol. 1998, 8, 101. (8) MacPhee, C. E.; Dobson, C. M. J. Mol. Biol. 2000, 297, 1203. (9) Jime´nez, J. L.; Guijarro, J.; Orlova, E.; Zurdo, J.; Dobson, C. M.; Sunde, M.; Saibil, H. R. EMBO J. 1999, 18, 815. (10) Rochet, J.; Lansbury, P. T. Curr. Opin. Struct. Biol. 2000, 10, 60. (11) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334. (12) Goda, S.; Takano, K.; Yamagata, Y.; Nagata, R.; Akutsu, H.; Maki, S.; Namba, K.; Yutani, K. Protein Sci. 2000, 9, 369. (13) Krebs, M. R. H.; Wilkins, D. K.; Chung, E. W.; Pitkeathly, M. C.; Chamberlain, A. K.; Zurdo, J.; Robinson, C. V.; Dobson, C. M. J. Mol. Biol. 2000, 300, 541.

Amyloid Fibril Formation of a Peptide (14) Nielson, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40, 6036. (15) Walsh, D. M.; Hartley, D. M.; Kusumoto, Y.; Fezoui, Y.; Condron, M. M.; Lomakin, A.; Benedek, G. B.; Selkoe, D. J.; Teplow, D. B. J. Biol. Chem. 1999, 274, 25945. (16) Pertinhez, T. A.; Bouchard, M.; Smith, R. A. G.; Doubson, C. M.; Smith L. J. FEBS Lett. 2002, 529, 193. (17) Li, S. C.; Deber, C. M. J. Biol. Chem. 1993, 268, 22975. (18) Chaloin, L.; Vidal, P.; Heitz, A.; VanMau, N.; Mery, J.; Divita, G.; Heitz, F. Biochemistry 1997, 36, 11179. (19) 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. (20) Remington, S. J.; Anderson, W. F.; Owen, J.; Ten Eyck, L. F.; Grainger, C. T.; Matthews, B. W. J. Mol. Biol. 1978, 18, 81. (21) Weaver, L. H.; Matthews, B. W. J. Mol. Biol. 1987, 193, 189. (22) Sayle, R. A.; Milner-White, E. J. Trends Biochem. Sci. 1995, 20, 374. (23) Najbar, L. V.; Craik, D. J.; Wade, J. D.; McLeish, M. J. Biochemistry 2000, 39, 5911. (24) Anderson, C. O.; Niesen, J. F. M.; Blanch, H. W.; Prausnitz, J. M. Biophys. Chem. 2000, 84, 177. (25) Anderson, C. O. Ph.D. Thesis, University of California, Berkeley, CA. (26) Najbar, L. V.; Craik, D. J.; Wade, J. D.; Salvatore, D.; McLeish, M. J. Biochemistry 1997, 36, 11525. (27) Naiki, H.; Higuchi, K.; Matsushima, K.; Shimada, A.; Chen, W. H.; Hosokawa, M.; Takeda, T. Lab. InVest. 1990, 62, 768.

Biomacromolecules, Vol. 5, No. 5, 2004 1823 (28) Levine, H. Protein Sci. 1993, 2, 404. (29) Tjernberg, L. O.; Callaway, D. J. E.; Tjernberg, A.; Hahne, S.; Lillieho¨o¨k, C.; Terenius, L.; Thyberg, J.; Nordstedt, C. J. Biol. Chem. 1999, 274, 12619. (30) Yang, Y.; Barker, S.; Chen, M. J.; Mayo, K. H. J. Biol. Chem. 1993, 268, 9223. (31) Albert, J. S.; Hamilton, A. D. Biochemistry 1995, 34, 984. (32) Liu, W.; Bratko, D.; Prausnitz, J. M.; Blanch. H. W. Biophys. Chem. 2004, 107, 289. (33) Llina´s, M.; Klein, M. P. J. Am. Chem. Soc. 1975, 97, 4731. (34) Thomas, P. D.; Dill, K. A. Protein Sci. 1993, 2, 2050. (35) Jasanoff, A.; Fersht, A. R. Biochemistry 1994, 33, 2129. (36) Bouchard, M.; Zurdo, J.; Nettleton, E. J.; Dobson, C. M.; Robinson, C. V. Protein Sci. 2000, 9, 1960. (37) de la Paz, M. L.; Goldie, K.; Zurdo, J.; Lacroix, E.; Dobson, C. M.; Hoenger, A.; Serrano, L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16052. (38) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Mcleish, T. C. B.; Nyrkova, I.; Radford, S. E.; Semenov, A. J. Mater. Chem. 1997, 7, 1135. (39) Aggeli, A.; Nyrkova, I. A.; Bell, M.; Harding, R.; Carrick, L.; Mcleish, T. C. B.; Semenov, A. N.; Boden, N. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 11857. (40) Lu, K.; Jacob, J.; Thiyagarajan, P.; Conticello, V. P.; Lynn, D. G. J. Am. Chem. Soc. 2003, 125, 639. (41) Schmittschmitt, J. P.; Scholtz, J. M. Protein Sci. 2003, 12, 2374.

BM049841E