Kinetically Controlled Thermal Response of β2-Microglobulin Amyloid Fibrils

doi:10.1016/j.jmb.2005.07.033

J. Mol. Biol. (2005) 352, 700–711

Kinetically Controlled Thermal Response of b2-Microglobulin Amyloid Fibrils Kenji Sasahara1, Hironobu Naiki2 and Yuji Goto1* 1

Institute for Protein Research Osaka University and CREST Japan Science and Technology Agency, Yamadaoka 3-2, Suita Osaka 565-0871, Japan 2

Faculty of Medical Sciences University of Fukui and CREST Japan Science and Technology Agency, Matsuoka, Fukui 910-1193, Japan

Calorimetric measurements were carried out using a differential scanning calorimeter in the temperature range from 10 to 120 8C for characterizing the thermal response of b2-microglobulin amyloid fibrils. The thermograms of amyloid fibril solution showed a remarkably large decrease in heat capacity that was essentially released upon the thermal unfolding of the fibrils, in which the magnitude of negative heat capacity change was not explicable in terms of the current accessible surface area model of protein structural thermodynamics. The heat capacity–temperature curve of amyloid fibrils prior to the fibril unfolding exhibited an unusual dependence on the fibril concentration and the heating rate. Particularly, the heat needed to induce the thermal response was found to be linearly dependent on the heating rate, indicating that its thermal response is under a kinetic control and precluding the interpretation in terms of equilibrium thermodynamics. Furthermore, amyloid fibrils of amyloid b peptides also exhibited a heating rate-dependent exothermic process before the fibril unfolding, indicating that the kinetically controlled thermal response may be a common phenomenon to amyloid fibrils. We suggest that the heating rate-dependent negative change in heat capacity is coupled to the association of amyloid fibrils with characteristic hydration pattern. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: amyloid fibril; thermal unfolding; b2-microglobulin; amyloid b peptide; hydration of fibrils

Introduction The specific self-association of proteins to form amyloid fibrils is an important process because amyloidoses have been recognized as conformational diseases that arise from the conversion of globular proteins into insoluble amyloid fibrils.1,2 This process involves nucleation coupled to the selfassociation step, which constitutes an alternative folding pathway to that leading to the native conformation.3,4 Amyloid fibril formation is now recognized as a generic phenomenon of proteins and peptides, regardless of the diverse structures of their native or initial states, suggesting that amyloid fibrils are formed by a common mechanism or that

Abbreviations used: Ab, amyloid b peptide; ASA, accessible surface area; b2-m, b2-microglobulin; CD, circular dichroism; Cp,app, apparent heat capacity; DSC, differential scanning calorimeter; Ts, starting temperature for the thermal unfolding of b2-m amyloid fibrils. E-mail address of the corresponding author: [email protected]

amyloidogenic intermediates with common structural properties are formed.5,6 b2-Microglobulin (b2-m), a 99 residue protein with a molecular mass of 11,800, is the light chain of the class I major histocompatibility complex (MHCI).7 In its native fold, b2-m adopts a typical immunoglobulin fold consisting of seven b-strands organized into two b-sheets linked by a single disulfide bridge.7 b2-m is also found as a major component of amyloid fibrils deposited in patients receiving long-term hemodialysis.8 Although the increase in b2-m concentration in blood over a long period is the most critical risk factor causing amyloidosis, the molecular details remain unknown. The relatively small size of b2-m makes it suitable for physicochemical studies addressing the amyloid fibril formation in the context of protein conformation.9–11 Although much effort has been devoted in recent years to elucidating the mechanism of b2-m amyloid fibril formation,12–19 the energetics and thermodynamics underlying amyloid fibril formation remain largely unclear. In the last few decades, calorimetric measurements by the differential scanning calorimeter

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

Thermal Stability of Amyloid Fibrils

(DSC) have been carried out extensively on the reversible folding/unfolding transition of globular proteins under equilibrium conditions.20–22 These studies have focused on the energetics of the transition between the folded and unfolded states, establishing the mechanisms of structural stabilization of proteins in terms of the equilibrium thermodynamics. We expect that calorimetric studies are promising to clarify the thermodynamics of amyloid fibril formation. Recently, Kardos et al.23 measured the enthalpy and heat capacity changes associated with b2-m amyloid fibril formation using isothermal titration calorimetry, suggesting a lower level of internal packing in comparison with the native conformation. Here, DSC measurements were carried out to monitor the process of the thermal response of b2-m amyloid fibrils. The obtained results give a clear example of the protein thermodynamics which significantly deviate from the monomolecular equilibrium transition established for small globular proteins, revealing a new aspect of the energetic basis of the structural stability of amyloid fibrils.

Results DSC thermogram of b2-m amyloid fibrils DSC measurements of b2-m amyloid fibril solutions in the temperature range from 10 to 120 8C at various fibril concentrations between 0.015 and 0.28 mg/ml (1.3 and 23.6 mM) showed unusual DSC thermograms, which accompany the thermal unfolding (i.e. depolymerization) of the fibrils (Figure 1(a)). To show the unique concentration dependence, the heat capacity values are not scaled by the protein concentration. For a comparison, the DSC thermogram of native b2-m at 0.125 mg/ml is presented, showing a single positive peak, which is magnified in Figure 1(b). It is noteworthy that the heat capacity of the amyloid fibrils at the same protein concentration (curve 5) was much smaller than that of the native state, leading to a large negative heat capacity, which is termed therefore apparent heat capacity (Cp,app). Compared to the case for b2-m with native tertiary structure, the heat capacity of b2-m amyloid fibrils exhibited a common temperature function that gradually became more negative as the temperature increased (w85 8C). Furthermore, when the fibril state was heated to 120 8C, the heat capacity increased drastically at around 60–100 8C, returning back to a value close to zero. As a result, a peak minimum was observed at around 60–90 8C. The reversibility of the DSC thermogram up to 120 8C was investigated (Figure 1(c)). The DSC thermogram in the second heating of the sample immediately after cooling from the first scan up to 120 8C corresponded to that of acid-unfolded b2-m monomer at the same pH 2.5, indicating that amyloid fibrils were thermally unfolded by the

701 first DSC scan. The reversibility of the DSC thermogram prior to the peak minimum was also investigated (Figure 1(c)). After one cycle of heating to the temperature just before this peak minimum and cooling down to 10 8C, the second thermogram showed the same trace as the first. Based on the results of these reheating runs, the starting temperature (Ts) for the thermal unfolding was defined at the peak minimum of the DSC thermogram. Additionally, the temperature range, in which the Cp,app value increases drastically at around 60–100 8C, corresponded to an abrupt transformation of the structure from the amyloid fibril to the thermally unfolded state, as shown by far-UV CD (see Figure 5 below). Thus, while the major transition starting at Ts and leading to the global unfolding of amyloid fibrils was irreversible, the heat effect prior to the unfolding, which we call “pre-transition”, was reversible. The fibril concentration dependence of the Cp,app value at 60 8C, which was obtained from the DSC thermograms observed at the various fibril concentrations, showed that the Cp,app value was saturated at a fibril concentration above 0.04 mg/ml (Figure 1(d)). An electrical thermal pulse for calibration of the calorimeter confirmed that the observed saturation was not caused by instrumental saturation. The results suggest that the decreases of Cp,app upon heating (i.e. pretransition) cannot be explained directly by the properties of proteins. The Ts values, the minima of DSC curves, were experimentally determined for various fibril concentrations ranging from 0.01 to 0.28 mg/ml. The plot of Ts against fibril concentration revealed an increase of 20 deg. C in the Ts value as the fibril concentration increased (Figure 1(e)). This result indicates that the amyloid fibrils shift towards a thermally more stable state as the fibril concentration increases. Kinetically controlled thermal response of b2-m amyloid fibrils DSC thermograms of b2-m amyloid fibrils at 0.1 mg/ml measured at various heating rates, ranging between 5 to 90 deg. C/h, revealed the distinct heating rate-dependence of the Cp,apptemperature curve: as the heating rate decreases, the pre-transition became less significant (Figure 2(a)). On the other hand, the Ts value increased with the decrease of the heating rate (Figure 2(b)). The hyperbolic curve suggests the complicated relation between Ts and heating rate. On the basis of the DSC thermograms as shown in Figure 2(a), the Cp,app values at 0.1 and 0.2 mg/ml at 67 8C, the temperature before the thermally unfolding of fibrils, were plotted as a function of the heating rate (Figure 2(c)). The Cp,app values at 67 8C, which were similar between the two b2-m concentrations, decreased linearly with the increase in the heating rate. To infer the equilibrium Cp,app value, the Cp,app values at 67 8C were extrapolated to a zero heating rate. The extrapolated Cp,app values

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Thermal Stability of Amyloid Fibrils

Figure 1. Thermal unfolding of b2-m amyloid fibrils measured by DSC at pH 2.5. (a) Representative DSC thermograms of b2-m amyloid fibril solutions. The b2-m amyloid fibril concentration was varied from 0.015 to 0.28 mg/ml; (1) 0.015, (2) 0.025, (3) 0.04, (4) 0.075, (5) 0.125, (6) 0.17, (7) 0.2, and (8) 0.28 mg/ml. The heating rate was 60 deg. C/h. For a comparison, the DSC thermogram of native b2-m at pH 7.0 is shown and magnified in (b), in which the b2-m concentration and heating rate were 0.125 mg/ml and 40 deg. C/h, respectively. (c) DSC thermograms of reheating of b2-m amyloid fibril solutions at 0.1 mg/ml b2-m, 60 deg. C/h, and pH 2.5. (1) Scan up to 120 8C. (2) Second scan after the first scan up to 120 8C. (3) Overlay of repeated scans up to 67 8C, showing complete reversibility. (4) Thermogram of the acid-unfolded b2-m. (d) Dependence of heat capacity (Cp,app) at 60 8C on amyloid fibril concentration. The Cp,app values at 60 8C were obtained from the DSC thermograms recorded at a rate of 60 deg. C/h as shown in (a). (e) The fibril concentration dependence of the starting temperature (Ts) for the global unfolding of b2-m amyloid fibrils. The Ts values were obtained from the DSC thermograms recorded at 60 deg. C/h as shown in (a).

corresponded to those of acid-unfolded b2-m within the experimental uncertainty, indicating that the difference between the equilibrium Cp,app values in the fibril states and those of the acid unfolded states is a hardly detectable change under these experimental conditions. To focus the significant decrease of the Cp,app value during the pre-transition, the reheating runs prior to Ts were carried out at various heating rates (Figure 3(a)). The second thermogram after cooling from the first run was almost the same as the first

run at any heating rate, confirming the reversibility of the pre-transition. The Cp,app values at 20 and 67 8C, which were obtained from Figure 3(a), decreased linearly as a function of the heating rate, consistent with the same plot constructed on the basis of the entire DSC thermograms including the global unfolding of fibrils (Figure 2(c)). When the respective Cp,app values obtained at 20 and 67 8C were extrapolated to the zero heating rate, the two Cp,app values corresponded with each other within the range of experimental uncertainty. This result

Thermal Stability of Amyloid Fibrils

703

Figure 2. Heating rate-dependence of the DSC thermograms of b2-m amyloid fibrils at pH 2.5. (a) Representative DSC thermograms of b2-m amyloid fibrils (0.1 mg/ml) at various heating rates. The heating rates were (1) 90, (2) 60, (3) 40, (4) 28, and (5) 25 deg. C/h. (b) Heating rate-dependence of Ts at 0.1 mg/ml b2-m amyloid fibrils. (c) The heating rate dependence of Cp,app at 67 8C at 0.1 (B) and 0.2 mg/ml (,) b2-m amyloid fibrils. The continuous lines show the linear fit of these data according to the least square method. Filled black circle and square at heating rate zero represent the Cp,app values of the acid-unfolded b2-m at 0.1 and 0.2 mg/ml at pH 2.5, respectively.

Figure 3. Analysis of heat-induced exothermic process of b2-m amyloid fibrils (0.1 mg/ml) at pH 2.5. (a) DSC thermograms from 10 to 68 8C at various heating rates. Heating was performed twice at each heating rate, showing the complete reproducibility of the exothermic process. The heating rates were (1) 15, (2) 20, (3) 30, (4) 40, (5) 50, (6) 60, (7) 70, (8) 80, and (9) 90 deg. C/h. (b) Heating rate-dependence of Cp,app values at 20 (B, C) and 67 (,, &) 8C. The Cp,app values were obtained from (a). Open and filled symbols represent the results of first and second scans, respectively. (c) Heating rate dependence of Qr value. The Qr values were calculated over the temperature range from 20 to 67 8C according to equation (4) and were plotted as the negative values (exothermic reaction).

indicates that the difference between the equilibrium Cp,app values (at the zero heating rate) at 20 and 67 8C is also small in comparison with the large heat effects of the pre-transition. The area of the exothermic process in Figure 3(a), i.e. the heat (Qr) needed to induce the kinetic thermal response, was calculated in the tempera-

ture range of 20 to 67 8C at the various heating rates according to equation (4) (see Materials and Methods) and was plotted as a function of the heating rate (Figure 3(c)). As expected from the above results, the Qr value was linearly dependent on the heating rate and extrapolated to zero at the zero heating rate.

704 The effect of NaCl on the thermal response of b2-m amyloid fibrils Figure 4(a) shows the DSC thermograms of b2-m amyloid fibrils at higher NaCl concentrations, where electrostatic shielding effects on the b2-m amyloid fibrils were expected. The Ts values significantly increased as the NaCl concentration

Thermal Stability of Amyloid Fibrils

was raised from 0.1 M to 1.0 M, indicating that the amyloid fibrils shifted towards a thermally more stable state as the NaCl concentration was increased. On the other hand, the addition of NaCl did not change notably the Cp,app temperature curve before the global unfolding. The reheating experiments confirmed the reversibility of the pretransition even in the presence of higher concentrations of NaCl. The Cp,app values at 20 and 67 8C in the presence of 1.0 M NaCl plotted against the heating rates (Figure 4(b)) showed basically the same dependences as those in 0.1 M NaCl (Figure 3(b)). Furthermore, the Qr values were calculated in the temperature range of 20 to 67 8C according to equation (4) (see Materials and Methods), showing that the Qr value decreases only slightly upon increasing the NaCl concentration (Figure 4(c)). Again, these plots demonstrate a good fit to a straight line against the heating rate, which approximately goes through zero at the zero heating rate. Light-scattering of b2-m amyloid fibril solutions Light-scattering was used to detect the heatinduced association (aggregation) of b2-m amyloid fibrils. Figure 5(a) shows the time-courses of lightscattering of b2-m amyloid fibril solutions at 0.2 mg/ ml during the heating process. The light-scattering intensity of fibril solutions increased slightly as the temperature rose from 20 to 64 8C at the heating rates of 63, 79, and 106 deg. C/h, suggesting the heatinduced weak association of b2-m amyloid fibrils. When the temperature of the fibril solution was raised from 20 to 64 8C at much higher heating rates of 147 and 264 deg. C/h, we observed visible aggregates of fibrils, which were reflected in the perturbation of light-scattering intensity. These results indicate that b2-m amyloid fibrils are susceptible to the heating rate-dependent association (aggregation). CD spectra of b2-m in various states

Figure 4. Effects of NaCl on the thermal response of b2-m amyloid fibrils (0.1 mg/ml) at pH 2.5. (a) DSC thermograms at various NaCl concentrations. Heating rate was 60 deg.C/h. The NaCl concentrations were (1) 0.1, (2) 0.2, (3) 0.3, (4) 0.4, (5) 0.5, (6) 0.6, and (7) 1.0 M. (b) Heating rate-dependence of Cp,app values at 20 (B, C) and 67 (,, &) 8C in the presence of 1.0 M NaCl. Heating was performed from 10 to 68 8C twice at each heating rate. Open and filled symbols represent the results of first and second scans, respectively. (c) Heating rate-dependence of Qr values at various NaCl concentrations. The Qr values were calculated over the temperature range from 20 to 67 8C according to equation (4) and plotted as the negative values. The NaCl concentrations were (C) 0.1, (&) 0.5, (:) 1.0, and (%) 1.5 M.

The far-UV CD spectra for the various states of b2-m are shown in Figure 5(b). The far-UV CD spectrum of b2-m at pH 7.0 showed a positive peak at 200 nm and a weak negative peak at 220 nm, as observed for b2-m in the native fold.12,14 The protein at pH 2.5 exhibited a somewhat negative ellipticity over the wavelength region of 250–200 nm, which is characteristic of a substantially acid-unfolded protein.12,14 The far-UV CD spectrum of b2-m amyloid fibrils formed at the same pH 2.5 demonstrated a large negative band centered on 218 nm, suggesting an increased amount of the b-sheet content.12,14 On the other hand, when these b2-m amyloid fibrils were heated to 120 8C at a rate of 60 deg. C/h, the sample after cooling showed almost the same far-UV CD spectrum as that of the acid-unfolded state at pH 2.5. This result indicates that b2-m amyloid fibrils after heating to 120 8C unfold into the acidunfolded state, which is consistent with the result from DSC measurement (Figure 1(c)).

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Thermal Stability of Amyloid Fibrils

To obtain additional information regarding fibril structural changes, we tried to construct the thermal unfolding transition curve of b2-m amyloid fibrils by following the negative ellipticity change at 218 nm as a function of increasing temperature. Although no significant change in the secondary structure of amyloid fibrils was observed before fibril unfolding (i.e. during the pre-transition), it was hard to determine the exact starting temperature (Ts) due to the slight aggregation of fibrils, which also causes the decrease in CD intensity. Instead, the b2-m amyloid fibril solutions (0.1 mg/ ml) prepared at 37 8C were incubated at each temperature of 25 to 96 8C for 5 minutes and after cooling the CD measurements of the fibril solutions were conducted at 20 8C. The CD intensity at 218 nm as a function of incubation temperature indicated that the starting temperature of thermal unfolding was approximately w80 8C (Figure 5(c)), corresponding to the result that b2-m amyloid fibrils have a Ts value of about 74 8C in the DSC measurements under the same solution conditions (Figure 1(c)). DSC thermograms of Ab amyloid fibrils

Figure 5. Thermal responses of b2-m amyloid fibrils measured by light-scattering (a) and CD ((b) and (c)). (a) The time-course of light-scattering of b2-m amyloid fibrils during the heating process at pH 2.5. The protein concentration was 0.2 mg/ml. The temperature was increased from 20 8C to 64 8C at the various heating rates of (1) 63, (2) 79, (3) 106, (4) 147, and (5) 264 deg. C/h. (b) Far-UV CD spectra for different conformational states of b2-m at 20 8C. (1) Native state at pH 7.0; (2) amyloid fibrils at pH 2.5; (3) acid-unfolded state at pH 2.5; and (4) after heating amyloid fibrils at pH 2.5 to 120 8C at 60 deg.C/h. The protein concentration was 0.1 mg/ml. (c) The change in the secondary structure of b2-m amyloid fibrils as a function of the incubation temperature at pH 2.5. The b2-m amyloid fibrils (0.1 mg/ml) prepared at 37 8C were incubated at each temperature of 25 to 96 8C for 5 min, and after cooling the CD measurements were conducted at 20 8C.

To examine whether the negative Cp,app change detected for b2-m amyloid fibrils before the fibril unfolding is common to other amyloid fibrils, DSC thermograms of amyloid fibrils of two types of amyloid b peptides, Ab(1–40) and Ab(25–35), were recorded. The molecular masses of Ab(1–40) and Ab(25–35) are 4330 and 1060, respectively. The Cp, app traces of Ab(1–40) amyloid fibrils revealed a remarkably large decrease in Cp,app as the temperature increased from 10 to 90 8C, followed by an abrupt increase in Cp,app (Figure 6(a)). In addition, the fibril concentration dependence of the Cp,app value (for example at 60 8C) was saturated at a fibril concentration above 0.09 mg/ml (20 mM), similar to the case of b2-m amyloid fibrils (Figure 1(d)). Similar behaviors were also observed with Ab(25–35) amyloid fibrils (data not shown). The reversibility of the Cp,app trace prior to the peak minimum was checked by reheating runs at the various heating rates for both Ab(1–40) and Ab(25– 35) amyloid fibrils. The second Cp,app trace after cooling from the first heating was virtually the same as the first at any heating rate (Figure 6(b) and (c)). The Cp,app values at 20 and 67 8C obtained from the Cp,app-temperature traces in Figure 6(b) and (c) were found to be linearly dependent against the heating rate (Figure 6(d) and (e)). The heat (Qr) values needed to induce the exothermic process in Figure 6(b) and (c) were calculated over the temperature range of 20 to 67 8C at the various heating rates according to equation (4). The resultant Qr values were linearly dependent on the heating rate (Figure 6(f)). Thus, thermal responses of Ab(1–40) and Ab(25–35) amyloid fibrils were kinetically controlled, as was the case of b2-m amyloid fibrils.

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Thermal Stability of Amyloid Fibrils

Figure 6. Kinetic thermal response of Ab amyloid fibrils at pH 7.2. (a) DSC thermograms of Ab(1–40) amyloid fibril solutions. The Ab amyloid fibril concentrations were varied from 0.02 to 0.17 mg/ml; (1) 0.02, (2) 0.04, (3) 0.07, (4) 0.09, (5) 0.13, and (6) 0.17. The heating rate was 60 deg. C/h. (b) and (c) Reversibility of Cp,app traces for amyloid fibril solutions of Ab(1–40) (b) and Ab(25–35) (c) from 10 to 76 8C at various heating rates. Heating was performed twice at each heating rate, showing the complete reproducibility of the exothermic process. Amyloid fibril concentrations were 0.09 mg/ml and 0.04 mg/ml for Ab(1–40) and Ab(25–35), respectively. The heating rates (deg. C/h) were (1) 20, (2) 30, (3) 40, (4) 50, (5) 60, (6) 70, (7) 80, and (8) 90. (d) and (e) Heating rate-dependence of Cp,app values at 20 (B, C) and 67 (,, &) 8C. The Cp,app values for Ab(1–40) (d) and for Ab(25–35) amyloid fibrils (e) were obtained from (b) and (c), respectively. Open and filled symbols represent the results of first and second scans, respectively. (f) Heating rate dependence of Qr value. The Qr values were calculated over the temperature range from 20 to 67 8C according to equation (4) and were plotted as the negative values (exothermic reaction). Red marks: Ab(1–40) amyloid fibrils (0.09 mg/ml); blue marks: Ab(25–35) amyloid fibrils (0.04 mg/ml). Red and blue lines were linearly best fitted to the respective data. The black line is for b2-m amyloid fibrils (0.1 mg/ml) and the same as that in Figure 3(c).

Discussion Unique heat capacity profile of b2-m amyloid fibril solution To address the thermodynamic stability of amyloid fibrils, we carried out the DSC measurements of b2-m amyloid fibrils. Unexpectedly, the

DSC diagrams of amyloid fibrils exhibited unique profiles distinct from those of the globular native state (Figure 1). With the increase in temperature up to 60–90 8C, the exothermic process occurs through the gradual decrease in Cp,app. Furthermore, temperature dependence of Cp,app becomes larger with the increase in the heating rate. This pre-transition detected by Cp,app is followed by the global unfolding of amyloid fibrils detected by both

Thermal Stability of Amyloid Fibrils

Cp,app and CD. Validity of the two-step process consisting of the pre-transition and major unfolding transition was confirmed by the reversibility of the pre-transition (Figure 3(a)) and also by the disappearance of characteristic Cp,app profile upon complete unfolding of amyloid fibrils at 120 8C (Figure 1(c)). These results argue that the thermal responses of amyloid fibrils are largely different from that of the globular native state. A unique heat capacity profile was also observed for Ab amyloid fibrils (Figure 6). Ab(1–40), which is the major component of the amyloid deposits associated with Alzheimer’s disease, is one of the most intensively studied amyloidogenic peptides.24 Ab(25–35) is a C-terminal fragment of Ab(1–40) which is assumed to form the central core stabilizing the Ab amyloid fibrils.25 The significance of the unique heat capacity profile is strengthened by the encouraging agreement between the results obtained for the three amyloid species, suggesting that the kinetic thermal response is common to amyloid fibrils. These heat effects cannot be explained by the models proposed by SanchezRuiz et al.,26 in which the reversible unfolding of monomeric state followed by the irreversible aggregation was assumed. The analysis of heat capacity changes has been of central importance in understanding the thermodynamics of protein folding, protein–protein binding, protein–nucleic acid binding, and the hydrophobic effect involved in these processes.27–31 Especially, heat capacities of hydration have become an important area of study because they significantly contribute to the heat capacity changes in the biological reactions described above.27–33 For example, the heat capacity change observed in protein unfolding is largely the result of changes in hydration of groups that are buried from the solvent in the native state, and has been successfully parameterized by the accessible surface area (ASA) to the solvent of the buried residues.20,21,34 In the case of b2-m with 99 residues, the heat capacity change upon unfolding is roughly estimated to be 5 kJ MK1 KK1 from the ASA model, which is close to the value of 5.6 kJ MK1 KK1 reported.23 These values can be transformed into the value of about 2.6!10K5 J KK1 per 0.125 mg/ml protein concentration used in the DSC measurement (Figure 1(a)). This value is too small and within the experimental uncertainty of the present DSC measurements (Figure 1(b)). Recently, the enthalpy and heat capacity changes of the b2-m fibril formation were estimated by isothermal titration calorimetry,23 indicating a similar heat capacity change upon amyloid formation (4.8 kJ MK1 KK1) to that of the folding to the native globular state, while the enthalpy change of the fibril unfolding (124 kJ MK1 at 37 8C) proved to be markedly lower than that of protein unfolding (175 kJ MK1 at 37 8C). In comparison with the native state, the results suggested the unique structural features of the amyloid fibrils: a similar extent of surface burial even with the supramolecular

707 architecture of amyloid fibrils, a lower level of internal packing, and the possible presence of unfavorable side-chain contributions.23 Even these thermodynamic parameters of fibril unfolding cannot explain the extensive kinetic effects observed here, although it is likely that they contribute partly to the observed effects. Transient fibril association and contribution of hydration to heat capacity Although the exact explanation remains unknown, there are a couple of features important for addressing the molecular mechanism of the unique Cp,app profiles. Firstly, the intensity of lightscattering of b2-m amyloid fibril solutions increases as the temperature increases (Figure 5(a)). Notably, an abrupt increase in the heating rate evidently induces visible aggregates of the fibrils. The visible aggregates were also observed for two Ab amyloid fibril solutions during the rapid heating process (data not shown). Therefore, it is likely that the decrease in Cp,app is related to the association or aggregation of fibrils (Figure 3(a)). Furthermore, the experimental facts that the Cp,app trace of the pretransition is heating rate-dependent and has a satisfactory reversibility suggest that amyloid fibrils transiently associate with each other during the heating process. The associated fibrils start to dissociate at Ts, resulting in the complete unfolding of amyloid fibrils at 120 8C. Calorimetric experiments have often revealed that the aggregation of thermally unfolded proteins proceeds with heat release, although the phenomena have not seriously been considered so far. Recently, Dzwolak et al.35 have reported that a significant decrease in heat capacity occurs in the aggregate (fibril) formation of bovine insulin, which shows the heating rate-dependent, exothermic process revealing features of nucleation-controlled kinetics. The exothermic process associated with a negative heat capacity change, which was observed for three amyloid fibril species in this study and for the aggregate formation of proteins, may have common significance for the molecular-level mechanism underlying the deposition of amyloid fibrils into various organs and tissues. The heat capacity effect of hydration is thought to be mainly attributable to the local changes in the thermally accessible motion modes, such as vibration, libration, and rotation, of bound water molecules.27,31,36 For instance, it is reported that, in addition to water molecules buried in the hydrophobic interface, bridging water molecules in a highly polar surface environment can also contribute substantially to the negative heat capacity change in the formation of a protein–DNA complex.31,36 Moreover, it is suggested that the buried water molecules in protein–protein interfaces have a lower heat capacity than bulk solvent and this burial of water molecules is reflected in the negative change in heat capacity for protein–protein association.30 A similar argument might be made

708 for the results obtained here. That is, the transient organization of fibril–water network, in which some of the water molecules are trapped upon the fibril– fibril association, might induce a reduction of the motion mode of these water molecules, reducing their heat capacity. Another important feature is that the negative Cp, app values in the pre-transition temperature (e.g. 60 8C) become constant above a particular concentration (0.04 mg/ml (3.4 mM) for b2-m and 0.09 mg/ ml (20 mM) for Ab(1–40), and 0.04 mg/ml (40 mM) for Ab(25–35)) (Figures 1(d) and 6(a)). It is noted that the critical concentrations in terms of weight concentration are similar to each other. This is of prime importance for the clarification of the thermal response of amyloid fibrils, because behavior like this has not been obviously observed in the DSC measurements of globular proteins. Although we cannot give a clear explanation for the unique concentration dependence of the heat effect, it is evident that the magnitude of negative heat capacity change is not explicable in terms of the current accessible surface area model of protein structural thermodynamics. This suggests that the observed negative Cp,app was caused by a large number of hydrated or trapped water molecules specific to amyloid fibrils. In accordance with this, amorphous aggregates b2-m prepared in the presence of high salt did not exhibit this large heat effect (data not shown). It is likely that excessive and extensive hydration of aggregated fibrils is sensitively reflected in the decrease of Cp,app. If this is the case, the saturation of heat capacity against the concentration of amyloid fibrils may represent saturation of the amount of hydrated water molecules. While hydration of the globular native state or solvated unfolded state has been studied intensively,20,21 the same is not true for the solvated water molecules of amyloid fibrils. Possible mechanism of the thermal stability under kinetic control On the basis of the above considerations, we suggest a possible mechanism explaining the kinetic heat effects on amyloid fibrils (Figure 7). There is increasing evidence suggesting that protein aggregation, including amyloid fibril formation, has been driven by hydrophobic interaction operating on proteins in an unfolded or partially unfolded state.37–39 The hydrophobic interaction is generally believed to be strongly temperature-dependent; hydrophobic interactions become stronger with the increase in temperature.20 In this context, as the heating rate increases, amyloid fibrils would transiently associate with each other due to the inter-fibrillar hydrophobic interaction. This association may be accompanied by more motionless bound water molecules, thus resulting in the decrease in the C p,app value with increasing temperature (w90 8C) (Figure 7). The significant increase in Ts with increasing NaCl concentration (Figure 4(a)) suggests the enhanced tendency of the

Thermal Stability of Amyloid Fibrils

Figure 7. The possible schemes for the heating ratedependence of the thermal response of b2-m amyloid fibrils. At lower heating rates, the intra-molecular hydrophobic interactions within the fibrils are enhanced with the increase in temperature, thus producing the stabilized fibrils with an increased Ts value. At higher heating rates, the inter-molecular hydrophobic interactions compete with the slow intra-molecular interactions, producing the associated fibrils. The associated fibrils are accompanied by the trapping of water molecules in fibril–fibril network and also by excessive hydration of clusters of exposed polar groups, thus resulting in the decrease in the Cp,app value. Increase of temperature up to 120 8C eventually unfolds both types of amyloid forms, producing the unfolded momomeric b2m. While the major transitions leading to the global unfolding are irreversible, pre-transitions are reversible.

inter-fibrillar interactions under the conditions of reduced electrostatic repulsion, consistent with the role of hydrophobic interactions. However, we do not know the clear reason of the absence of NaClconcentration dependence of the Cp,app value in the pre-transition region (Figure 4(a)). Recent studies have shown that b2-m amyloid fibrils exhibit a range of morphologies with different numbers of protofilaments, and the final fibril states arise through the superhelical twists of protofibrils, with possible structural rearrangements.40,41 The lateral association of fibrils as suggested in Figure 7 might be related to the formation of a variety of aligned fibrils as observed in tissue deposits. At lower heating rates, the possible structural rearrangement of amyloid fibrils may be induced due to intra-fibrillar hydrophobic interactions without excess bound water molecules, resulting in the increase in Ts (Figure 2(b)). In the light of the experimental fact that the common kinetic thermal response was observed for three amyloid species despite their different sidechains (Figure 6(f)), we suggest that the kinetic effects are related to the fundamental properties of fibril structure such as specific arrangement of the hydrophobic and hydrogen-bonding residues. The native structures of globular proteins are optimized so that the hydrogen bonds are surrounded by the hydrophobic side-chains, producing maximal contributions of hydrophobic interaction and hydrogen bonds to protein stability.42 On the other hand,

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Thermal Stability of Amyloid Fibrils

upon the formation of amyloid fibrils dominated by main-chain interactions,6 it is conceivable that defects of hydrophobic wrapping (i.e. exposed hydrogen bonds and polar residues) exist in various regions of the molecule and also on the surface of the fibrils, separately from the hydrophobic clustering. This separate clustering of hydrophobic and polar groups may induce an intrinsic potential to form excessive hydration around the clusters of polar groups. The aggregation of amyloid fibrils upon rapid heating might further enhance the separate clustering of hydrophobic and polar groups, leading to the remarkable kinetic heat effects: the heating rate-dependent large decrease in heat capacity.

Conclusion The thermal responses of b2-m and Ab amyloid fibrils are under kinetic control with enormous amplitude of negative heat, precluding the interpretation of calorimetric data in terms of the structural thermodynamics. Although the exact mechanism remains unknown, the data suggest that heating rate-dependent negative change in heat capacity is mainly attributed to the transient organization of fibril–water network, which is produced by the fundamental properties of amyloid fibrils forming cross-b sheet structure. It is conceivable that the heating rate-dependent large decrease in heat capacity is coupled with the excessive hydration of polar group clusters transiently formed in aggregated amyloid fibrils. Studies of non-equilibrium states are important, because many systems in vivo exist as nonequilibrium states.43 So far the lack of experimental evidence has limited detailed discussion on the physical significance of the dynamics in nonequilibrium states. The results obtained here suggest that the amyloid fibril system provides an excellent model to investigate the kinetically controlled thermal response of protein and peptide molecules. To understand the observed heat effects, it will be important to study directly the properties of hydrated or trapped water molecules.

Materials and Methods b2-m amyloid fibril formation Recombinant human b2-m was expressed in Escherichia coli and purified as described.17 b2-m amyloid fibrils were prepared by the fibril extension method established by Naiki and co-workers,9,10 in which sonicated b2-m amyloid fibril seeds (final concentration: 5 mg/ml) were extended with monomeric protein (0.3 mg/ml) in 50 mM citrate buffer containing 100 mM NaCl (pH 2.5).23 The seed fibrils were prepared by the repeated extension reaction of purified b2-m amyloid fibrils from patients at pH 2.5.9,15 The protein solution in the presence of the seed fibrils was incubated for 3 h at 37 8C without agitation in a temperature-controlled incubator and the formation of

b2-m amyloid fibrils with a needle-like morphology was confirmed by thioflavin T binding and atomic force microscopy. Almost all the monomeric b2-m was converted to amyloid fibrils under this condition.23 The amyloid fibril solutions were diluted with the buffer (50 mM sodium citrate (pH 2.5) containing 100 mM NaCl) to the desired concentration of fibrils for the measurement, in which the fibril states were stable to dilution. In the DSC measurement at higher NaCl concentration, the amyloid fibril solutions were diluted with the buffer (250 mM sodium citrate, pH 2.5) and 2.5 M NaCl solution. The monomer concentrations of b2-m were determined spectrophotometrically at 280 nm using the extinction coefficient of 1.63 ml mgK1 cmK1.17 Ab amyloid fibril formation Ab(1–40) and Ab(25–35) were purchased from Peptide Institute, Inc. (Osaka, Japan). Ab(1–40) was dissolved in a 0.02( (w/v) ammonia solution at 2.17 mg/ml (500 mM) at 4 8C. Mature Ab(1–40) amyloid fibrils were prepared by the fibril extension method, in which the fibrils fragmented by sonication (i.e. seed fibrils, final concentration 4.3 mg/ml) were extended with the monomeric peptides (173 mg/ml) in the polymerization buffer (50 mM sodium phosphate buffer (pH 7.2), containing 100 mM NaCl) overnight at 37 8C.44 The seed fibrils were prepared by the repeated extension reaction.44 Ab(25–35) was first dissolved in dimethylsulfoxide at 5.3 mg/ml (5 mM) and then diluted with deionized water to 0.53 mg/ml (500 mM). Mature Ab(25–35) amyloid fibrils (final concentration: 63.6 mg/ml or 60 mM) were spontaneously formed in the polymerization buffer overnight at 37 8C.44 The amyloid fibril solutions were diluted with the polymerization buffer to the desired concentration of fibrils for the measurement, in which the fibril state was stable to dilution. Calorimetric measurements Calorimetric measurements were carried out using a Microcal VP-DSC calorimeter (Northampton, MA) within the temperature range of 10–120 8C under excess pressure of 25 psi. Sample and buffer solutions at pH 2.5 were properly degassed in an evacuated chamber for 3 min at 25 8C and then carefully loaded into the sample and reference cells (operating volume w0.5 ml), respectively. The buffer solution without the protein was 50 mM sodium citrate containing NaCl (0.1–1.5 M). A background scan corrected with buffer solution in both sample and reference cells was subtracted from the scan of the sample and buffer solutions. Apparent heat capacity (Cp, app) corresponding to the whole sample solution was recorded using ORIGIN software (Microcal Inc.). Light-scattering measurements Light-scattering measurements were carried out with a Hitachi fluorescence spectrometer, model F-4500, using a quartz cell with a light path of 10 mm. The slit lengths for excitation and emission were 1.0 and 1.0 nm, respectively, and the wavelengths were both set at 350 nm. The temperature of the fibril solutions was controlled by circulating water from a thermostat. CD measurements CD measurements were conducted at 20(G0.1) 8C

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Thermal Stability of Amyloid Fibrils

using a 1 mm cell by an AVIV model 215s spectropolarimeter equipped with a thermoelectrically controlled cell holder (AVIV Association, Lakewood, NJ). CD spectra for the various states of b2-m were recorded in the far-UV region from 250 to 200 nm using a 1 mm cell, with a step size of 0.5 nm and an average time of 20 s. CD intensity at 218 nm of b2-m amyloid fibril solution was recorded every 10 s for 2 minutes with a response time of 8 s and averaged. CD data of the appropriate buffers were recorded and subtracted from those of protein.

Data analysis The heat capacity change (DCp,org) for the heating ratedependent exothermic process of fibril–water system may be defined as follows: DCp;org ðTÞ Z Cp;app ðTÞKCp;0 ðTÞ

(1)

where Cp,app(T) and Cp,0(T) are the heat capacities of the fibril solution in the kinetic exothermic process and at equilibrium state (at zero heating rate), respectively. The Cp,0(T) value corresponds to the Cp,app value extrapolated to the zero heating rate and is approximated to be constant in the temperature range from 20 to 67 8C on the basis of the results in Figures 3(b), 4(b) and 6(d) and (e). The Cp,app(T) value obtained from the experimental DSC thermogram of amyloid fibril solutions can be well approximated by: Cp;app ðTÞ Z Cp;app ðT1 Þ C aðTKT1 Þ C bðTKT1 Þ2

(2)

where a and b are constants, and T1 is a reference temperature. Then: DCp;org ðTÞ Z Cp;app ðT1 Þ C aðTKT1 Þ C bðTKT1 Þ2 KCp;0 ðTÞ

(3)

Then, the heat needed to induce the exothermic process of fibril–water system (Qr) is represented in the temperature range from T1 to T2 by: ð Qr ðTÞ Z DCp;org ðTÞdT Qr Z ðCp;app ðT1 ÞKCp;0 ÞðT2 KT1 Þ C a=2ðT2 KT1 Þ2

(4)

C b=3ðT2 KT1 Þ3 In the calculation of the Qr values, T1 and T2 are set at 20 8C (293.15 K) and 67 8C (340.15 K), respectively. The Cp,app values extrapolated to the zero heating rate at 67 8C were used as Cp,0. With a non-linear least-squares fitting program, the calculated DSC curves were fitted to the observed curves so that a and b were determined.

Acknowledgements This work was supported in part by grants-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science and Technology on Priority Areas (no. 40153770) and Scientific Research (B) (no. 13480219).

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Edited by K. Kuwajima (Received 21 April 2005; received in revised form 6 July 2005; accepted 11 July 2005) Available online 27 July 2005