Fibrillation in Human Serum Albumin Is Enhanced in the Presence of

Jul 20, 2010 - Andrea Stirpe , Manuela Pantusa , Bruno Rizzuti , Maria P. De Santo , Luigi Sportelli , Rosa Bartucci , Rita Guzzi. International Journ...
0 downloads 0 Views 1MB Size
Download PDF
10228

J. Phys. Chem. B 2010, 114, 10228–10233

Fibrillation in Human Serum Albumin Is Enhanced in the Presence of Copper(II) Nitin K. Pandey,† Sudeshna Ghosh,† and Swagata Dasgupta* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India ReceiVed: April 29, 2010; ReVised Manuscript ReceiVed: June 28, 2010

The aggregation process in proteins is governed by several factors such as temperature, pH, presence of electrolytes, denaturants, and metal ions. Here, we report the role of Cu(II) in inducing rapid fibrillation in human serum albumin. We have monitored this process via UV-vis spectroscopy, fluorescence spectroscopy, circular dichroism, ζ-potential measurements, electron paramagnetic resonance studies, fluorescence microscopy, and field emission scanning electron microscopy. Images show a fibrillar network of human serum albumin in the presence of Cu(II) in 60% ethanol incubated at 65 °C at physiological pH. All other studies also support the enhanced fibrillation in presence of Cu(II). Introduction Proteins and peptides undergo self-aggregation leading to the formation of oligomers with different morphology in vitro under specific biophysical conditions. This pathway may in turn lead to the formation of amyloid fibrils, which appear to be a generic feature of polypeptide chains.1–3 The fibrillar morphology associated with amyloid fibrils is linked to various neurological disorders, such as Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases.4,5 Protein misfolding, a stochastic event causing these diseases, generates fibers not only in the case of disease proteins but also for disease-unrelated proteins.6 Recent studies have reported the coordination of metal ions with amyloid fibrils where Cu(II) has been purported to be central to amyloid-β (Aβ) neurotoxicity.7–9 Copper, an essential element, plays a critical role in human metabolism and exists in the body in either a chelated form or bound to proteins. Apart from a role in the modulation of the fibrillation pathway, Cu(II) has been shown to induce aggregation of proteins. Cu(II) is known to bind to several amyloid-forming proteins and peptides and demonstrates accelerating and/or inhibiting effects on the fibrillation process depending on the protein and biophysical conditions.10–14 Human serum albumin (HSA), a natively R-helical protein (>60%) with 17 disulfide bridges, consists of three domains, I, II, and III, each with two subdomains (A and B).15,16 HSA plays an important role in the transportation of fatty acids, metal ions, and other physiologically important compounds. Under normal circumstances, native HSA is reluctant to form fibers due to its stabilized helical structure.17 The formation of HSA fibrils thus requires a necessary destabilization to generate partially folded intermediates that aggregate to form fibrils.18 This can be accomplished by lowering the pH, increasing the temperature, and adding salts, denaturants, and metal ions. Conditions under which fibrils are formed in HSA include ethanol concentrations varying from 40 to 60% and also incubation at ambient temperature after incubation at 65 °C for 6 h.18 Information is available on the conformational transitions in HSA in the presence of aqueous ethanol solutions.19 It has been reported that the aggregation process does not require nucleation and incubation of HSA solutions at varying pH and ionic strength, resulting in the formation of different supramolecular assemblies * To whom correspondence should be addressed. Tel: +91 3222 283306. Fax: +91 3222 255303. E-mail: [email protected]. † Both authors contributed equally to the work.

of HSA amyloid-like fibers.20,21 The pH and ionic strength have also been shown to play a role in the steps leading to HSA fibrillation with the observation that fibril formation is affected by electrostatic shielding.22 The interaction of Cu(II) with HSA has been investigated earlier, but no reference has been made to how this may affect the fibrillation process. HSA is known to coordinate Cu(II), which plays an important role in its transportation and metabolism in vivo.23 Specific binding sites in the protein have also been identified, which includes an N-terminal metal binding site and also a multimetal binding site.24,25 HSA has been reported to show picomolar affinity to Cu(II) at its N-terminal site, with the local level of HSA being projected as an important factor in regulating the availability of Cu(II) to pathological targets.26 Amino acid residues such as His act as potential ligands for Cu(II), and it has been shown that binding essentially occurs at a single site in HSA.27 The complex thus formed involves the R-NH2 terminus, NH of Ala2, and NH of His3 as also its imidazole nitrogen. Considering a protein to metal ratio of 1:1, we have investigated the effect of Cu(II) on the fibrillation process of HSA. The effect of Cu(II) on the fibrillation of HSA has been investigated under conditions that require an initial destabilization followed by incubation at ambient temperature. The destabilization was achieved by taking the protein and metal ion in a ratio of 1:1 in 60% ethanol. This was followed by incubation at 65 °C at pH 7.4 for solutions of HSA with and without Cu(II). The choice of 65 °C arises from the fact that there exists a two-state transition (Tm values ∼56 °C and ∼62 °C) that occurs during the denaturation of HSA corresponding to the sequential unfolding of the domains as mentioned in ref 20, where a Tm value of 66 °C has also been reported.28,29 This temperature is chosen to ensure partial unfolding of HSA that facilitates aggregation. HSA fibrils were obtained in the solutions with and without Cu(II) when heated at 65 °C in the presence of ethanol at pH 7.4 followed by incubation at room temperature. The solutions were monitored over time by Congo Red (CR)-based UV studies, thioflavin T (ThT) fluorescence studies that are characteristic of fibril formation in addition to circular dichroism (CD) experiments, ζ-potential measurements, and electron paramagnetic resonance (EPR) studies. Images of the fibers were obtained through fluorescence microscopy and field emission scanning electron

10.1021/jp103876p  2010 American Chemical Society Published on Web 07/20/2010

Fibrillation in Human Serum Albumin

J. Phys. Chem. B, Vol. 114, No. 31, 2010 10229

Figure 1. (a) Time evolution of ThT intensity for HSA and HSA-Cu(II) during the initial heating phase at 65 °C at pH 7.4 in 60% ethanol with protein and dye concentrations at 1 and 2 µM, respectively. (b) Histogram of periodic change in ThT intensity for HSA and HSA-Cu(II) solutions over a period of ∼240 days.

Figure 2. (a) Change in secondary structure composition (measurement at 208 nm) for HSA and HSA-Cu(II) within the initial period of heating at 65 °C up to 6 h at pH 7.4 in the presence of 60% ethanol. (b) Far UV CD spectra of HSA-Cu(II) solutions over a period of 120 days, indicating an increase in mdeg value with incubation time. Inset: Difference in millidegree values of HSA-Cu(II) and HSA at 208 nm (∆mdeg) with time. (c) CD spectra of HSA-Cu(II) and HSA after 60 days.

microscopy (FE-SEM). Preliminary observations indicate a faster rate of fibrillation in solutions containing Cu(II) in comparison to solutions without Cu(II). The importance of the present study lies in an understanding of the aggregation process of HSA in the presence of metal ions. Despite conditions that are not associated with any pathological situation, it is nevertheless interesting to note the effect of Cu(II) on HSA fibril formation. A recent study has investigated cellular effects of bovine serum albumin fibrils and studied the signaling cascade induced by the fibrillar protein where the fibrils were found to induce apoptosis.30 Materials and Methods Materials. HSA, ThT, and CR were purchased from Sigma Chemical Co. (St. Louis, MO) and used as received. Copper sulfate pentahydrate (CuSO4 · 5H2O) and all other chemicals used were from SRL (India).

Preparation of Fibrils. HSA was dissolved in doubledistilled water, and the concentration was measured spectrophotometrically at 280 nm using a molar extinction coefficient of 35219 M-1 cm-1.31 Stock solutions were prepared keeping the protein concentration at 160 µM for each set at an ethanol concentration of 60% (v/v) at pH 7.4. In the case of Cu(II) sets, the HSA to Cu(II) ratio was kept as 1:1 (160 µM). For each analysis, phosphate buffer of pH 7.4 was used for dilution of samples. Fibrils were obtained by heating each set at 65 °C for 6 h followed by incubation at room temperature. ThT Fluorescence. The change in ThT intensity for the kinetic studies during the initial hours of heating was acquired by quenching extracted aliquots at different time intervals at 0 °C. After samples attained room temperature, ThT was added, and the samples were incubated for 5 min and scanned in a Jobin Yvon Fluoromax 4 Spectrofluorimeter. For other measurements over a longer period of time, aliquots from protein

10230

J. Phys. Chem. B, Vol. 114, No. 31, 2010

Pandey et al. required staining and placed on a glass slide covered with a coverslip and monitored by means of a Leica DM 2500 M microscope equipped with a fluorescence attachment. Filter cube no. 2 (Leica I3 11513878, BZ: 01) was used for ThT excitation and emission. The images were acquired with a Leica DFC 310 FX camera attached with the microscope. All observations were performed at 10X/0.25 (N PLAN EPI). Results and Discussion

Figure 3. ζ-Potential measurements monitored for the initial period of heating at 65 °C for HSA solutions with and without Cu(II) in the presence of 60% ethanol at pH 7.4.

Figure 4. EPR spectra of (X-band, 9.13 GHz) of a frozen solution of the [HSA-Cu(II)] system incubated at pH 7.4 at 60% ethanol heated at 65 °C for 6 h followed by incubation at room temperature.

solutions with and without Cu(II) were taken and diluted to a final concentration of 1 µM with phosphate buffer, keeping the ThT concentration at 2 µM in the working solutions. Samples were incubated for 10 min prior to recording measurements. Excitation and emission wavelengths were set at 450 and 482 nm, respectively, keeping the slit width at 5 nm and an integration time of 0.3 s. All spectra were corrected with respect to the corresponding blank. CD. Far-UV CD spectra were recorded on a JASCO-810 automatic recording spectrophotometer. Quartz cuvettes with a 0.1 cm path length were used. CD spectra were accumulated at 25 °C at a scan rate of 50 nm/min between 190 and 240 nm. Aliquots drawn at different time intervals were diluted with phosphate buffer to maintain a final concentration of 5 µM for each solution. The protein secondary structure content was determined using the online DICHROWEB server.32 ζ-Potential Measurement. ζ-Potential was measured at 25 °C with a Zetasizer Nano ZS (Malvern Instruments). Each measurement was performed on a freshly extracted aliquot of the protein sample and diluted with phosphate buffer to a final concentration of 8 µM at different time intervals. The data reported are the means ( standard errors of at least four different measurements. EPR Spectroscopy. Incubated samples were taken in a standard quartz EPR tube and frozen at 77 K. EPR spectra of the samples were recorded on a JEOL -JES-FA 200, 9.13 GHz spectrometer. Fluorescence Microscopy. Five microliters of 1 mM ThT was mixed with 10 µL of each solution set to achieve the

Kinetics of Fibrillation. Fibrillation of HSA was characterized by both CR and ThT, both of which are able to bind with fibers due to the inherent core β-sheet structural motif of fibers.33,34 ThT is an amyloid-specific dye as it does not fluoresce on excitation at 450 nm under normal circumstances but, upon binding with amyloid fibrils, shows significant emission maxima around 482 nm.35 The fluorescence intensity at 482 nm has been compared for HSA solutions incubated with and without Cu(II). Initial kinetic studies as shown in Figure 1a indicate that the extent of formation of HSA fibrils is slightly more in the presence of Cu(II) in comparison to samples without Cu(II). This enhancement gets more pronounced with time as indicated in Figure 1b. Even though we observe a ∼40% rise in fibril formation for HSA solutions as compared to a ∼30% rise in the case of those with Cu(II) for the initial 30 days of incubation at room temperature, the greater intensity in the case of the latter under the same conditions is indicative of a greater degree of fibril formation. With progression in fibril formation, the growth rate decreases for HSA but is clearly more rapid in the case of solutions with Cu(II). The data shown in Figure 1a were subjected to a nonlinear least-squares curve fitting analysis using a stretched exponential function given by F ) F∞ + ∆F exp[-(ksp*t)n], where F, F∞, and ∆F correspond to the observed fluorescence intensity at time t, the final fluorescence intensity and fluorescence amplitude, respectively, and ksp represents the rate of spontaneous fibril formation.36 This approach has also been used to study of the effect of electrostatic interactions on HSA fibrillation and in the study of the kinetics of amyloid fibril formation of β-lactoglobulin in the presence of urea.22,36 The n and ksp values obtained for HSA are 1.1 ( 0.24 and 0.34 ( 0.038 h-1, respectively, and those for HSA-Cu(II) are 1.90 ( 0.36 and 0.24 ( 0.015 h-1, respectively. The relatively higher n value in the case of HSA-Cu(II) as compared to HSA is indicative of a greater cooperative phenomena in the case of HSA-Cu(II). This result is in agreement with the other studies that suggest that Cu(II) facilitates HSA fibrillation. We observe the characteristic red shift (∼40 nm) for both cases with CR binding that is indicative of fibril formation.33 The difference in absorption between CR bound HSA and HSA-Cu(II) fibrillar species is attributed to the formation of CR binding species. The results obtained for HSA fibril formation match well with previous studies on the protein20 (Figure S1 in the Supporting Information). Effect on Secondary Structure. Far UV-CD spectra of HSA and HSA-Cu(II) solutions indicate a gradual loss in R-helical structure and a corresponding rise in the β-sheet content on fibril formation.37,38 CD spectra of HSA solutions prior to being subjected to treatment with 60% ethanol and heated to 65 °C show two minima at 208 and 222 nm, characteristic of the protein.39 In general, observations for solutions that have been treated to form fibrils show that there is a gradual disappearance of the minima at 222 nm with a reduction at 208 nm.20 During the initial heating at 65 °C for 6 h, the corresponding millidegree value at 208 nm increases (becomes less negative) for both HSA and HSA-Cu(II), indicating a decrease in helicity. The effect

Fibrillation in Human Serum Albumin

J. Phys. Chem. B, Vol. 114, No. 31, 2010 10231

Figure 5. Fluorescence microscopy images of HSA fibrils in the presence and absence of Cu(II) incubated at pH 7.4 at 60% ethanol heated at 65 °C for 6 h followed by incubation at room temperature: (a) HSA, 2 days; (b) HSA-Cu(II), 2 days; (c) HSA, 9 days; (d) HSA-Cu(II), 9 days; (e) HSA, 58 days; (f) HSA-Cu(II), 58 days; (g) HSA, 150 days; and (h) HSA-Cu(II), 150 days.

on the secondary structure of HSA is more marked in the presence of Cu(II) (Figure 2a). As expected, the spectra show a regular decrease in helical content with a concomitant increase in the β-sheet structure. A quantitative estimation of the changes in the relative R-helix and β-sheet content during the initial heating period indicates an overall ∼44 and ∼53% loss of R-helix structure for HSA and HSA-Cu(II), respectively. The rise in the β-sheet content is greater than twice the initial values in both cases (2.6 and 2.7, respectively). The results indicate a similar trend in both cases with significant helix to sheet transformation, as is expected on fibrillation with a comparatively greater disruption in the presence of Cu(II) (Table S1 and Figure S2 in the Supporting Information). A further progressive decrease in the helical content in solutions incubated with Cu(II) over a period of 120 days is

shown in Figure 2b that point toward a distinct loss in helical structure in comparison to the native form of the protein. The inset shows the relative increase in the millidegree value for solutions incubated with Cu(II). A representative CD spectra demonstrating the relative difference in ellipticity for the same period of time for solutions incubated with and without Cu(II) is shown in Figure 2c. In general, it is observed that incubation with Cu(II) leads to further disruption in the secondary structure content. The minima around 215-220 nm associated with β-sheet formation are not very apparent in the observed spectra. This observation is similar to reported studies that indicate that the minima become less prominent as a result of scattering caused by larger aggregated particles (fibrils) present in solution.22

10232

J. Phys. Chem. B, Vol. 114, No. 31, 2010

ζ-Potential Measurements. It is possible to measure progressive aggregation by the observing changes in the ζ-potential, the potential at the shear plane, since the total surface charge is affected during the aggregation process. A decrease in the ζ-potential is indicative of aggregation as shown in the case of concanavalin A.40 We have monitored the change in ζ-potential during the initial period of heating at 65 °C by removing aliquots from the HSA solutions incubated with and without Cu(II). Solutions incubated with Cu(II) show a greater reduction in the ζ-potential that is indicative of a greater propensity to aggregate. A plausible explanation may be that the partially unfolded protein that is negatively charged at a pH of 7.4, the pI of HSA being 4.9, tends to aggregate due to the exposure of inner hydrophobic domains in the protein. The presence of the metal ion facilitates the process by bringing the negatively charged moieties together, thereby resulting in a further lowering of the ζ-potential as shown in Figure 3. In the later period of incubation, the ζ-potential achieved a constant value of -21.8 ( 0.22 and -21.75 ( 0.26 mV for HSA and HSA-Cu(II), respectively, which indicates that there is no further significant change in electrostatic interaction. EPR Studies. To determine the oxidation state of copper during the fibrillation process, EPR spectra of different [HSA-Cu(II)] samples were obtained. The [HSA-Cu(II)] EPR spectrum (Figure 4) clearly depicts a single oxidized copper, that is, copper is present in the +2 state, during its interaction with HSA in the fibrillation process. There is normal parallel hyperfine splitting in the 270-300 mT region and rhombic splitting due to the splitting of the perpendicular region peaks around 300-350 mT. We observe four hyperfine signals for Cu(II), which are distinctly different from that of Cu(II) bound to native HSA where only two hyperfine signals with single Cu(II) species are reported.27 From the hyperfine splitting pattern, it appears that there is the possibility of the existence of two types of Cu(II) species41,42 with the hyperfine coupling constant A| (expressed in units of magnetic field) and g| (Lande g factor) values of 16.36 ( 1 mT and 2.26, respectively, for one type and A| and g| values of 21.12 ( 1 mT and 2.157, respectively, for the other. The g| value for the second type of Cu(II) is almost similar to the g| value (2.162) of Cu(II) bound to native HSA, which may be attributed to a similar type of coordination as in the case of native HSA.27 Further investigation is underway to understand the specific coordination involved. Change of Fibrillar Morphology over Time: Microscopic Images. The assemblies formed due to the process of aggregation and fibril formation show varied morphology ranging from spherical structures to elongated fibrils and also flat ribbonlike structures. External conditions of pH, temperatures are key factors responsible for the differential morphology observed in case of HSA.20 The presence of fibrils and its morphology during the span of incubation was studied via fluorescence microscopy and electron microscopy. A green fluorescence was observed when the ThT-stained fibrils were excited at near-UV wavelength (blue excitation) using an appropriate filter. Fluorescence images initially show threadlike fibers in the case of HSA, whereas the presence of Cu(II) initiates branching. On further incubation, a complex network was observed in the presence of Cu(II) in comparison to HSA only where scattered fibers were observed. Figure 5a,c shows thin fibrils within 10 days in both HSA and HSA in the presence of metal ion (Figure 5b,d) and indicates the initiation of branching. In the later period after the onset of branching, a distinct network formation is observed in the presence of Cu(II) ion (Figure 5f,h), whereas the HSA fibrillar morphology in the absence of Cu(II) does not change

Pandey et al. much (Figure 5e,g). FE-SEM images of HSA and HSA-Cu(II) after 12 days of incubation show a distinct curl in fiber growth in the presence of Cu(II), whereas scattered threadlike fibrils are obtained in the absence of Cu(II) (Figure S3 in the Supporting Information). Conclusion The importance of HSA as a carrier protein along with its aggregation propensity makes it a good target for studies relating to protein aggregation and fibril formation. Following the idea that a partial destabilization is required for this process to occur, we monitored the progress of fibril formation for HSA solutions in the presence of Cu(II). Our preliminary studies indicate that fibrillation is enhanced in the presence of Cu(II). Solutions have been monitored by ThT fluorescence, CD measurements, and ζ-potential studies. Aggregation is favored initially as indicated by the decrease in ζ-potential, and enhancement of fibril formation is evident from ThT fluorescence intensity measurements and also through fluorescence microscopic images. Initial ThT kinetic studies show that in presence of Cu(II), the increase in ThT intensity is greater, which is also supported by the CD kinetic study where a pronounced conformational change is observed. Fluorescence microscopy shows that at the preliminary level, threadlike fibers are formed in HSA and HSA-Cu(II), whereas at longer incubation times, the metal ion induces formation of a curly fibrillar network. Further work is in progress to assess the details of the fibrillation pathway and the effect of Cu(II) and other metal ions on the aggregation pathway. These findings will facilitate a further understanding of the aggregation of proteins in the presence of metal ions that have recently been shown to play a significant role in research relating to neurodegenerative diseases. Acknowledgment. S.D. is thankful to the Department of Science and Technology, Government of India, for support (SR/ SO/BB-54/2007). We are also thankful to Central Research Facility, IIT Kharagpur, for providing experimental facilities. N.K.P. and S.G. thank IIT Kharagpur and CSIR, New Delhi, respectively, for their fellowship. Supporting Information Available: CR experiment, figures and table for quantitative estimation of secondary structures (CD data), table for apparent rate calculated from different techniques, and FE-SEM images. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Dobson, C. M. Philos. Trans. R. Soc. London B 2001, 356, 133– 145. (2) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329–332. (3) Gazit, E. Angew. Chem., Int. Ed. 2002, 41, 257–259. (4) Dobson, C. M. Nature 2003, 426, 884–890. (5) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353–356. (6) Guijarro, J. I.; Sunde, M.; Jones, J. A.; Campbell, I. D.; Dobson, C. M. Proc. Natl. Acad. Sci. 1998, 95, 4224–4228. (7) Sarell, C. J.; Syme, C. D.; Rigby, S. E. J.; Viles, J. H. Biochemistry 2009, 48, 4388–4402. (8) Faller, P.; Hureau, C. Dalton Trans. 2009, 1080–1094. (9) Pradines, V.; Stroia, A. J.; Faller, P. New J. Chem. 2008, 32, 1189– 1194. (10) Atwood, C. S.; Moir, R. D.; Huang, X.; Scarpa, R. C.; Bacarra, N. M. E.; Romano, D. M.; Hartshorn, M. A.; Tanzi, R. E.; Bush, A. I. J. Biol. Chem. 1998, 273, 12817–12826. (11) Paik, S. R.; Shin, H. J.; Lee, J. H.; Chang, C. S.; Kim, J. Biochem. J. 1999, 340, 821–828. (12) Zou, J.; Kajita, K.; Sugimoto, N. Angew. Chem., Int. Ed. 2001, 40, 2274–2277. (13) Uversky, V. N.; Li, J.; Fink, A. L. J. Biol. Chem. 2001, 276, 44284– 44296.

Fibrillation in Human Serum Albumin (14) Stirpe, A.; Rizzuti, B.; Pantusa, M.; Bartucci, R.; Sportelli, L.; Guzzi, R. Eur. Biophys. J. 2008, 37, 1351–1360. (15) He, X. M.; Carter, D. C. Nature 1992, 358, 209–215. (16) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153–203. (17) Gorinstein, S.; Caspi, A.; Rosen, A.; Goshev, I.; Zemser, M. J. Pept. Res. 2002, 59, 71–78. (18) Taboada, P.; Barbosa, S.; Castro, E.; Mosquera, V. J. Phys. Chem. B 2006, 110, 20733–20736. (19) Taboada, P.; Barbosa, S.; Castro, E.; Gutierrez-Pichel, M.; Mosquera, V. Chem. Phys. 2007, 340, 59–68. (20) Juarez, J.; Taboada, P.; Mosquera, V. Biophys. J. 2009, 96, 2353– 2370. (21) Juarez, J.; Taboada, P.; Lopez, S. G.; Cambon, A.; Madec, M. B.; Yeates, S. G.; Mosquera, V. J. Phys. Chem. B 2009, 113, 12391–12399. (22) Juarez, J.; Lopez, S. G.; Cambon, A.; Taboada, P.; Mosquera, V. J. Phys. Chem. B 2009, 113, 10521–10529. (23) Appleton, D. W.; Sarkar, B. J. Biol. Chem. 1971, 246, 5040–5046. (24) Bal, W.; Christodoulou, J.; Sadler, P. J.; Tucker, A. J. Inorg. Biochem. 1998, 70, 33–39. (25) Sokołowska, M.; Krezel, A.; Dyba, M.; Szewczuk, Z.; Bal, W. Eur. J. Biochem. 2002, 269, 1323–1331. (26) Rozga, M.; Sokolowska, M.; Protas, A. M.; Bal, W. J. Biol. Inorg. Chem. 2007, 12, 913–918. (27) Valko, M.; Morris, H.; Mazr, M.; Telser, J.; McInnes, E. J. L.; Mabbs, F. E. J. Phys. Chem. B 1999, 103, 5591–5597. (28) Farruggia, B.; Rodriguez, F.; Rigatuso, R.; Fidelio, G.; Pico, G. J. Protein Chem. 2001, 20, 81–89.

J. Phys. Chem. B, Vol. 114, No. 31, 2010 10233 (29) Flora, K.; Brennan, J. D.; Baker, G. A.; Doody, M. A.; Bright, F. V. Biophys. J. 1998, 75, 1084–1096. (30) Huang, C.-Y.; Liang, C.-M.; Chu, C-Li.; Liang, S.-M. BMC Biotechnol. 2009, 9, 2, DOI: 10.1186/1472-6750-9-2. (31) Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein Sci. 1995, 4, 2411. (32) Whitmore, L.; Wallace, B. A. Nucleic Acids Res. 2004, 32, W668– W673. (33) Klunk, W. E.; Pettegrew, J. W.; Abraham, D. J. J. Histochem. Cytochem. 1989, 37, 1273–1281. (34) Levine, H. Protein Sci. 1993, 2, 404–410. (35) LeVine, H. Methods Enzymol. 1999, 309, 274–284. (36) Hamada, D.; Dobson, C. M. Protein Sci. 2002, 11, 2417–2426. (37) 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, 25945–25952. (38) Kirkitadze, M. D.; Condron, M. M.; Teplow, D. B. J. Mol. Biol. 2001, 312, 1103–1119. (39) Dockal, M.; Carter, D. C.; Ruker, F. J. Biol. Chem. 2000, 275, 3042–3050. (40) Vetri, V.; Canale, C.; Relini, A.; Librizzi, F.; Militello, V.; Gliozzi, A.; Leone, M. Biophys Chem. 2007, 125, 184–190. (41) Karr, J. W.; Szalai, V. A. J. Am. Chem. Soc. 2007, 129, 3796– 3797. (42) Chattopadhyay, M.; Walter, E. D.; Newell, D. J.; Jackson, P. J.; Spencer, E. A.; Peisach, J.; Gerfen, G. J.; Bennett, B.; Antholine, W. E.; Millhauser, G. L. J. Am. Chem. Soc. 2005, 127, 12647–12656.

JP103876P