Pressure-Accelerated Dissociation of Amyloid Fibrils in Wild-Type Hen Lysozyme

Abstract

The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml⁻¹ fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (k_obs = 0.00193 min⁻¹ at 0.1 MPa, 0.0348 min⁻¹ at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹ and the activation compressibility Δκ^‡ = −0.0075 ± 0.0006 ml mol(monomer)⁻¹ bar⁻¹ for the dissociation reaction. The negative ΔV^0‡ and Δκ^‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril.

Introduction

Amyloidosis is a group of diseases that are caused by the formation of amyloid fibrils and are still difficult to prevent or cure (1). Once amyloid fibrils are formed, they dissociate extremely slowly, if at all, at ambient pressure, which severely limits our ability to study the dissociation mechanism of amyloid fibrils in physicochemical terms in vitro. Moreover, although a number of reports have shown that pressure dissociates aggregates or amyloid fibrils (2, 3, 4, 5, 6, 7, 8), other studies have shown that pressure has a limited effect on dissociation (9) or even accelerates the formation of amyloid fibrils (10, 11). Therefore, we consider it important to characterize the dissociation reaction of amyloid fibrils as physicochemical processes of protein molecules that follow a stoichiometry of reaction. Our previous studies on the disulfide-deficient mutant (0SS) showed that the dissociation reaction of the protofibril is greatly accelerated by pressure (12, 13, 14). A detailed study using high-pressure fluorescence showed a negative activation volume and a negative activation compressibility for the dissociation reaction of the 0SS protofibril (14), consistent with the finding that the amyloid fibril is in a state of high volume and compressibility (15).

Here, we report a kinetic analysis of the pressure dissociation reaction of amyloid fibrils from a naturally occurring (wild-type) protein from hen lysozyme. Under physiological conditions, hen lysozyme, a single-chain protein with 129 residues, folds into a stable conformer that is rich in α-helices and has four disulfide bridges. To increase the tendency for fibril formation, in this work we used low pH and high temperature instead of mutation to reduce its stability (16). One can efficiently transform hen lysozyme at low pH into amyloid fibrils in vitro by seeding it with preformed fibrils at elevated temperatures (17, 18, 19, 20, 21). We applied high-pressure fluorescence to study the dissociation reaction of amyloid fibrils from wild-type hen lysozyme. On atomic force microscopy (AFM) images, the amyloid fibrils from wild-type hen lysozyme are fairly thick (∼5 nm) and straight (Fig. 1), in contrast to the fibrils from 0SS, which are thin (∼2 nm) and curvy. Also, wild-type hen lysozyme can form bundles of protofibrils in maturation, whereas 0SS remains in protofibrils even after prolonged incubation.

Figure 1.

Amyloid fibrils of hen lysozyme as monitored by AFM. An AFM image of fibrils formed in the third generation (see Materials and Methods) is shown. The average height of the fibril (considered to represent the diameter of the fibril) is 5.2 ± 0.7 nm.

To our knowledge, there has been no detailed report on the kinetics of dissociation of amyloid fibrils from naturally occurring proteins. In this study, we were interested in determining whether pressure accelerates the dissociation of amyloid fibrils from wild-type hen lysozyme in a similar manner as in its intrinsically denatured mutant 0SS, i.e., whether they have a significant negative activation volume and a considerable negative activation compressibility for dissociation. This would give us insight into how generally and how effectively pressure can accelerate dissociation of amyloid fibrils, and in what range of pressure the acceleration is most effective. The fluorescence emissions from six Trp residues (Trp-28, -62, -63, -108, -111, and -123) of hen lysozyme are a sensitive reporter of the dissociation reaction. AFM reports the morphology of fibrils in considerable detail, whereas NMR identifies dissociated species.

Materials and Methods

Chemicals

Lyophilized hen egg white lysozyme (crystallized six times) was purchased from Seikagaku (Tokyo, Japan) and used without further purification. Other chemicals were reagent grade and obtained from Nakaraitesque (Kyoto, Japan).

Preparation of seed-induced amyloid fibrils

Amyloid fibrils consisting of intact wild-type hen lysozyme were prepared as described previously (17). Briefly, the lyophilized powder of wild-type hen lysozyme was dissolved to a final concentration of 8.0 mg/ml in water containing 80 mM NaCl with pH adjusted to 2.2 by HCl, and the solution was incubated at 57°C, the transition temperature of hen lysozyme (16). After ∼11 days of incubation, the resultant amyloid fibrils were subjected to extensive sonication to produce oligomers, an aliquot of which (10% w/w) was mixed into a fresh aqueous solution of intact hen lysozyme (90% w/w, 8 mg ml⁻¹ in 80 mM NaCl at pH 2.2) and incubated for 4.5 h at 57°C. The second-generation fibrils produced in this way were subjected to extensive sonication to produce seeds. A fresh solution of intact hen lysozyme (96% w/w, 8 mg ml⁻¹ in 80 mM NaCl at pH 2.2) was mixed with the seeds (4% w/w) and incubated for 10 h at 57°C to produce third-generation fibrils consisting almost entirely of intact hen lysozyme molecules (Fig. 1).

AFM

We obtained AFM images at 0.1 MPa at 25°C in the cyclic contact mode at a frequency of 119 kHz on an SPI-3800 microscope (Seiko Instruments, Japan). We carried out the measurements by depositing an aliquot of fibril solutions on freshly cleaved mica surface. After an incubation of ∼1 min, we washed the mica a few times with pure water and gently dried it by blowing compressed nitrogen onto the surface.

Trp fluorescence measurements under pressure

For Trp fluorescence measurements, ∼100 μl of the fibril solution, freshly prepared by 100-fold dilution of the original fibril solution to 80 μg ml⁻¹ in 80 mM NaCl, pH 2.2, was transferred into a quartz cell housed in a high-pressure chamber (Syn, Kyoto, Japan) connected to an external high-pressure pump (Techno, Hiroshima, Japan). The high-pressure chamber was set into a fluorescence spectrometer (FP-6500; JASCO, Tokyo, Japan) for fluorescence measurements under variable pressure. Approximately 10 min after the dilution was completed, the pressure was set to a desired value. After ∼5 min, the first spectrum was measured, and then the spectrum was measured continuously every 2 min until the measurement was finished. During each measurement, the pressure was maintained with water as the pressure mediator to a fixed value between 3 and 450 MPa (1 MPa = 10 bar), and the temperature was maintained by circulating water through the high-pressure chamber at 25°C. Six Trp residues of hen lysozyme provided a sensitive probe for the conformational state of this protein. The excitation of Trp fluorescence was made at 295 nm with a slit width of 5 nm, and the fluorescence emission was collected from 300 nm to 450 nm (maximum intensity ∼340 nm) with a slit width of 10 nm.

¹H NMR measurement

¹H NMR spectra were measured at 0.1 MPa at 25°C on a Bruker AVANCE 600 spectrometer operating at 600 MHz.

Analysis of time-dependent fluorescence data

At each pressure the change in fluorescence intensity at a fixed wavelength is plotted against time, and the data points I(t) can be fitted with an exponential function of time,

$$\mathrm{I}\left(\mathrm{t}\right)=\left({\mathrm{I}}_0-{\mathrm{I}}_{\infty }\right)\text{exp}\left(-k_{\mathrm{obs}}\mathrm{t}\right)+{\mathrm{I}}_{\infty },$$ (1)

or alternatively with

$$\text{ln}\left[\frac{\left(\mathrm{I}\left(\mathrm{t}\right)-{\mathrm{I}}_{\infty }\right)}{\left({\mathrm{I}}_0-{\mathrm{I}}_{\infty }\right)}\right]=-k_{\mathrm{obs}}\mathrm{t},$$ (2)

where I(t) is the fluorescence intensity at time t, I₀ is the initial fluorescence intensity, and k_obs is the observed rate constant for dissociation averaged over the fibrils. Here, I_∞ is the fluorescence intensity at infinite time, assumed to correspond to full dissociation. Because we used the same concentration of fibrils for all measurements at different pressures, we assumed that I_∞ was the same at all pressures and was given from I_∞ at 400 MPa after 4 h, which attained nearly full dissociation of the fibrils. Then the I − I_∞ values were best-fitted to Eq. 2 to give k_obs at each pressure. The observed rate constant k_obs represents the rate of decay of the total mass of fibrils into the total mass of monomers. Furthermore, under the excessive dilution (100-fold dilution of the original fibril solution to 80 μg ml⁻¹), the association reaction was sufficiently slow, so that the observed relaxation rate constant k_obs may be considered to represent the dissociation process only.

Analysis of pressure-dependent dissociation rate

We analyzed the dissociation reaction of the hen lysozyme fibrils into hen lysozyme monomers by using the transition-state theory, in which ΔG^‡ determines the dissociation rate. The pressure-dependent Gibbs energy of activation, ΔG^‡, is given to the second-order in pressure P:

$$\Delta {\mathrm{G}}^{‡}=\Delta {\mathrm{G}}^{0‡}+\left(\Delta {\mathrm{V}}^{0‡}\right)\left(\mathrm{P}-{\mathrm{P}}^0\right)-\left(\frac{\Delta {\kappa }^{‡}}{2}\right){\left(\mathrm{P}-{\mathrm{P}}^0\right)}^2.$$ (3)

Then the rate constant (k) of the dissociation reaction can be expressed as

$$\text{ln}k=\text{ln}\left(\frac{k_{\mathrm{b}}T}{\mathrm{h}}\right)-\frac{\Delta {\mathrm{G}}^{0‡}}{\mathrm{RT}}-\left(\frac{\Delta {\mathrm{V}}^{0‡}}{\mathrm{RT}}\right)\left(\mathrm{P}-{\mathrm{P}}^0\right)+\left(\frac{\Delta {\kappa }^{‡}}{2\mathrm{RT}}\right){\left(\mathrm{P}-{\mathrm{P}}^0\right)}^2.$$ (4)

Further, we have the relations

$${\left(\partial \frac{\left(\text{ln}k\right)}{\partial \mathrm{P}}\right)}_{\mathrm{T}}=-\left(\frac{\Delta {\mathrm{V}}^{0‡}}{\mathrm{RT}}\right)+\left(\frac{\Delta {\kappa }^{‡}}{\mathrm{RT}}\right)\left(\mathrm{P}-{\mathrm{P}}^0\right)$$ (5)

$$\Delta {\mathrm{V}}^{‡}=\Delta {\mathrm{V}}^{0‡}-\Delta {\kappa }^{‡}\left(\mathrm{P}-{\mathrm{P}}^0\right)$$ (6)

$$\Delta {\kappa }^{‡}=-{\left(\frac{\partial \Delta {\mathrm{V}}^{‡}}{\partial \mathrm{P}}\right)}_{\mathrm{T}},$$ (7)

where k_b is the Boltzmann constant, h is the Planck constant, R is the universal gas constant, T is the absolute temperature, ΔG^0‡ is the Gibbs energy difference at 0.1 MPa, ΔV^‡ is the activation volume at pressure P, ΔV^0‡ is the activation volume at 0.1 MPa, and Δκ^‡ is the activation compressibility (assumed to be independent of pressure). Fitting the experimentally obtained k to Eq. 4 is expected to determine the activation volume ΔV^0‡ and the activation compressibility Δκ^‡ for the fibril dissociation.

On the other hand, the observed rate constant k_obs represents the rate of decay of only the total mass of fibrils that are heterogeneous in length to the mass of monomers, and not the rate constant of the stoichiometric reaction for dissociation. In reality, the dissociation reaction of each fibril is considered to be brought about by the successive steps of the fibrillation/dissociation reaction represented by

$$\mathrm{F}\left(n\text{-}\mathrm{mer}\right)\underset{k_{+}}{\overset{k_{\mathrm{-}}}{\rightleftarrows }}\mathrm{F}\left(n\text{-}1\text{-}\mathrm{mer}\right)+\text{monomer}$$ (8)

in which one monomer is added to or dissociated from one end of the fibril (14, 22). F(n-mer) and F(n-1-mer) represent fibrils consisting of n and n-1 monomers, respectively. Here we assume that k₊ and k₋ are the intrinsic rate constants for association and dissociation, respectively, and are independent of the length or the polymerization number (n) of the fibril. Then the observed rate constant k_obs is related to the intrinsic dissociation rate constant k₋ of each step of Eq. 8 by the relation (14)

$$k_{\mathrm{obs}}=k_{-}\left(1-r\right),$$ (9)

where r (<1) is the ratio of [F(n-1-mer)]/[F(n-mer)] (14). We note from Eq. 9 that the volumetric properties obtained from the pressure dependence of k_obs represents those of the intrinsic dissociation rate constant k₋ of the reaction represented by Eq. 8, which is common to all fibrils of different lengths.

Results

Morphology of fibrils as observed by AFM

Fig. 1 shows an AFM image of the amyloid fibrils of hen lysozyme as formed in the third generation (see Materials and Methods), which depicts fairly straight fibrils in contrast to the curvy ones for fibrils of its disulfide-deficient mutant 0SS (12). Moreover, we found that the average diameter of the fibril for hen lysozyme (given as the height of the fibril in the AFM image) is 5.2 ± 0.7 nm, which is distinctly larger than the ∼2 nm found for 0SS (14). However, the consistency of the diameter within the range of ±0.7 nm suggests that the fibrils are essentially protofibrils, a linear (helical) array of protein molecules, rather than matured fibrils, which would form bundles of protofibrils and therefore would give a variety of diameters. In contrast to the uniformity of the diameter, the length of the protofibril varies greatly from ∼100 nm to ∼2000 nm, showing a large heterogeneity in the number of monomer units that form a protofibril.

Pressure-accelerated dissociation of amyloid fibrils monitored by Trp fluorescence

At 0.1 MPa, the wavelength of maximum emission of the fluorescence from the fibril solution was found at ∼339 nm, which is fairly close to the ∼334 nm for native hen lysozyme, showing that the six Trp side chains are largely buried in the solvent-inaccessible regions of the fibril. When a high pressure was applied to the fibril solution, the fluorescence intensity started to decrease with time, whereas the change in maximum wavelength of emission remained marginal (Fig. 2). This is in contrast to the case of fibrils from intrinsically denatured mutant 0SS, in which the maximum wavelength of emission shifts from ∼338 nm to ∼350 nm (14). The decrease in fluorescence intensity indicates that the quantum yield of the Trp fluorescence becomes smaller upon dissociation, and the observation of no distinct red-shift of the fluorescence maximum indicates that the dissociated protein is not unfolded. These observations are consistent with the notion that the fibrils are dissociated into natively folded monomers for which fluorescence from Trp residues other than Trp-62 and -108 is largely quenched (23). The inset in Fig. 2 shows the decay of fluorescence intensity with time at 340 nm.

Figure 2.

Pressure-induced changes in the Trp fluorescence spectrum of hen lysozyme fibrils at 380 MPa at 25°C. Inset: Plot of the fluorescence intensity at 340 nm against time. The fibril solution, prepared at 8 mg ml⁻¹, was diluted to 80 μg ml⁻¹ in 80 mM NaCl, pH 2.2, before pressure was applied. The first spectrum was measured at ∼5 min after the pressure was set at 380 MPa, and the subsequent spectra were obtained at 2-min intervals for up to 140 min.

Fig. 3 gives the result of plotting data such as those shown in Fig. 2 at various pressures according to Eq. 2. Here we focus the time range restricted to the relatively early phase of the dissociation reaction (t < ∼60 min). This is because, as the average length of the fibrils becomes shorter, some species of hen lysozyme other than protofibrils (e.g., oligomers) may contribute to the dissociation, for which Eq. 8 would no longer hold. The plots of the data in Fig. 3 show straight lines at all pressures measured at least up to ∼60 min, with the slope of each line giving a single observed dissociation rate constant (k_obs) that increases considerably with pressure.

Figure 3.

Plot of logarithms of normalized fluorescence intensity at 340 nm, ln((I – I_∞)/(I₀– I_∞)), against time at 25°C at various pressures (data taken from Fig. 2).

In fact, the rate constant k_obs does not depend on time, at least for the initial ∼60 min (Fig. 3), which is consistent with the assumption that the dissociation reaction follows Eq. 8 with an intrinsic dissociation rate constant irrespective of the fibril length. It would be good if we could evaluate the intrinsic rate constant from Eq. 9. To do so, we would need to evaluate the r-value by noting an exponential distribution of fibril population against the fibril length on AFM (14). Unfortunately, we could not obtain a reliable length distribution of fibrils for the present system, probably because the fibrils contain a small fraction of bundle structures (i.e., matured fibrils) even though they consist mainly of protofibrils (cf. Fig. 1).

NMR evidence for dissociation into monomers

¹H NMR spectra measured on fibrils and fibrils pressure-treated at 400 MPa are shown in Fig. 4. A comparison of the spectra with that of intact hen lysozyme makes it unequivocally clear that the fibrils are dissociated into hen lysozyme monomers by pressure treatment. The result is consistent with the view that the reaction follows the linear polymerization mechanism of Eq. 8.

Figure 4.

¹H NMR spectra (600 MHz) of hen lysozyme under different conditions measured at 0.1 MPa, 25°C, pH 2.2. The spectral intensities are normalized for the concentration of hen lysozyme (0.4 mg/ml). (A) Fibrils as formed. (B) Fibrils after application of pressure at 400 MPa for 4 h at 25°C. (C) Intact hen lysozyme monomer.

Activation volume and activation compressibility

Fig. 5 shows the plot of the logarithm of k_obs against pressure in the range of 3–450 MPa. Extrapolation of the data to 0.1 MPa gives k_obs = 0.00193 min⁻¹ at 0.1 MPa, and k_obs = 0.0348 min⁻¹ at 400 MPa, showing an increase in the dissociation rate by 18 times. On the other hand, the logarithm of k_obs increases almost linearly at low pressure but becomes distinctly nonlinear above ∼200 MPa, reaching a plateau at ∼350–450 MPa. The initial increase in ln k_obs in the low-pressure range shows that ΔV^0‡ is negative, whereas the approach to a plateau in the high-pressure range shows that Δκ^‡ is negative. Fitting the data to Eq. 4 gives the values for activation volume and activation compressibility to be ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹ and Δκ^‡ = −0.0075 ± 0.0006 ml mol(monomer)⁻¹bar⁻¹ (or −0.52 × 10⁻⁶ ml g⁻¹bar⁻¹), respectively (Table 1). The retardation of acceleration at high pressure arises because ΔV^‡ is nulled due to the negative second term Δκ^‡ (P − P⁰) in Eq. 6. Although ΔV^0‡ and Δκ^‡ values were previously reported for the disulfide-deficient mutant 0SS (14), to our knowledge, we are the first to determine them in a naturally occurring protein.

Figure 5.

Plot of the logarithm of the observed dissociation rate constant (k_obs) against pressure. Values of k_obs were obtained from the data in Fig. 3. The solid line represents the least-squares fit of Eq. 4 to the data points, giving ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹ and Δκ^‡ = − 0.0075 ± 0.0006 ml mol(monomer)⁻¹bar⁻¹. Extrapolation to 0.1 MPa yields k_obs = 0.00193 min⁻¹ or τ = 8.6 h at 0.1 MPa, and k_obs = 0.0348 min⁻¹ or τ = 29 min at 400 MPa.

Table 1.

Activation volume and activation compressibility for dissociation of amyloid fibrils from hen lysozyme

Volumetric parameters
Hen lysozyme	ΔV0‡(ml mol−1)	Δκ‡ (ml mol−1 bar−1)
		(ml g−1 bar−1)
Wild-type∗	−32.9 ± 1.7	−0.0075 ± 0.0006
		(−0.5 × 10−6)
Mutant (0SS)†	−50.5 ± 1.6	−0.013 ± 0.001
		(−0.9 × 10−6)

^∗From the present experiment (80 mM NaCL, pH 2.2, 25°C).

^†Data taken from Abdul Latif et al. (14); pH and buffer conditions: sodium acetate buffer, pH 4.0, containing 30 mM NaCL, 25°C.

The negative ΔV^0‡ and Δκ^‡ values support the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and suggest that the transition involves a hydration-mediated activated state of the fibril. The negative signs for both ΔV^0‡ and Δκ^‡ values were also obtained previously for the mutant of hen lysozyme, 0SS (Table 1), showing that the above notion prevails at least in amyloid fibrils from hen lysozyme and related proteins.

Discussion and Conclusions

In Fig. 6 we provide a schematic volume diagram of the fibril and transition state of hen lysozyme based on our current results. The volume situation is quite different between the low-pressure (<200 MPa) and high-pressure (∼450 MPa) ranges. In the low-pressure range, a difference in volume between the fibril state and the transition state (ΔV^‡ ∼ ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹) renders pressure to dramatically accelerate the dissociation of amyloid fibrils from hen lysozyme. On the other hand, in the high-pressure range (∼450 MPa), ΔV^‡ becomes almost null because of the significant contribution of Δκ^‡ (= −0.0075 ± 0.0006 ml mol(monomer)⁻¹bar⁻¹) in the second term of Eq. 6. As a consequence, the dissociation rate shows a plateau at ∼450 MPa and is predicted to decrease at still higher pressures.

Figure 6.

Illustration of a volume diagram for the fibril state and the transition state of hen lysozyme at 25°C. At 0.1 MPa, the volume of the transition state is lower than that of the fibril state by ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹, but the volume difference becomes almost null at 450 MPa (Eq. 6). This is because the compressibility is larger for the fibril state than for the transition state (Δκ^‡ = −(∂ΔV^‡/∂P)_T = − 0.0075 ± 0.0006 ml mol(monomer)⁻¹bar⁻¹; Eq. 7), indicating that the fibril state is more compressible than the transition state. Note that because the volume difference between the monomer and the fibril state is not exactly known, their levels are arbitrarily drawn.

At 0.1 MPa, a relatively large difference in volume between the fibril state and the transition state (ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹) is consistent with the notion that the fibril has a relatively high volume, which becomes smaller in the transition state. Abdul Latif et al. (14) observed this situation in 0SS (ΔV^0‡ = −50.5 ± 1.60 ml mol(monomer)⁻¹, although their absolute values are different from ours. The high-volume nature of the fibril was verified for the protofibril of 0SS directly by independent density measurement at 0.1 MPa (15). The high-volume states of the fibrils are considered to arise commonly from packing defects (cavities) and/or salt bonds.

On the other hand, the negative Δκ^‡ (= −0.0075 ± 0.0006 ml mol(monomer)⁻¹bar⁻¹) means that the fibril state is more compressible than the transition state. Previous ultrasonic velocity measurements for 0SS experimentally showed a high compressibility of the fibril state (15). Although we were unable to perform such measurements for our system due to more-stringent experimental conditions, such as high temperature (57°C), we may expect a similarly high compressibility from the high-volume state of the fibril, as discussed above, with packing defects likely to be present. When the packing defects of the fibril are decreased in the transition state by partial hydration, the transition state will become less compressible, resulting in a negative Δκ^‡ as observed.

The smaller ΔV^0‡ and Δκ^‡ values for wild-type hen lysozyme compared with the 0SS mutant appear to be related to the distinct difference in morphology of the two fibrils, i.e., the relatively straight protofibril with an apparent diameter of 5.2 ± 0.7 nm (Fig. 1) versus the windy protofibril with an apparent diameter of ∼2.0 nm in the latter (12, 14). On the other hand, the lower volume of the transition state (negative ΔV^0‡) suggests that hydration takes place in the transition state. Hydration and packing have been considered to be important factors that characterize amyloid fibrils (24). Our results confirm that notion by showing that they may play key roles in the dissociation of amyloid fibrils from hen lysozyme and its mutant.

The dynamics of amyloid fibrils, including their formation and dissociation, may be of fundamental importance in life. Although the phenomenon of pressure-accelerated dissociation has been widely recognized in amyloid fibrils from various sources of protein, the volumetric properties of this dissociation have not been well documented (2, 3, 4, 5, 6, 7, 8, 9). The volumetric concept and kinetic dissociation experiments, such as that shown in Fig. 6, could provide a useful framework for designing pressure-related experiments to elucidate the dynamics of amyloid fibrils, as well as for dissociating amyloid fibrils by pressure in future studies.

Editor: Shin'ichi Ishiwata.